During the science trip where Peter is bitten, Harry says that the microscope is the "largest electron microscope on the eastern seaboard". However, the microscope images shown are taken from a phase contrast microscope not an electron microscope. (Phase contrast gives less magnification but lets you work with live material, for example, the cell being manipulated in the image shown on the film).
When the name "Green Goblin" is first suggested, Mr. Jameson says something like "Call the Patent Office, I want to copyright that name." This confuses two different departments. The PTO (Patent and Trademark Office) deals only in Patents and Trademarks, while copyrights are issued by the Copyright Office. He should have said either "Call the Patent Office, I want to trademark that name," or "Call the Copyright Office, I want to copyright that name."
When Peter is fighting the carjacker in the warehouse, the thief drops his pistol to throw a knife at Peter, but when Peter thows him against the wall, the pistol is suddenly back in the car jacker's hand.
During the World Unity Festival, when the Green Goblin throws his pumpkin bomb at the balcony full of board members, as the windows blow up, Harry can be seen running away from Mary Jane and toward the area he will later have his head hit. In the next shot, Harry is seen still standing next to Mary Jane.
The morning after the field trip, as Peter is following Mary Jane, we see the car that is going to pick her up drive past Peter. When we switch to Peter's point of view, the car is nowhere to be seen. Then we go back to Peter, the bus drives up, and the car suddenly appears.
During the parade attack sequence when MJ falls from the balcony she is first seen falling side-down. As Spider-Man catches her she is falling face-down. In the next shot, of Spider-Man about to catch her from above, she is on her side again.
When Peter is testing out his newfound web powers in his room, he uses target practice to pick up a Dr. Pepper can. In two quick shots, we see his light bulb lamp is turned off; however, when he finally manages to hit the can, the light bulb lamp is turned on.
When the general is talking to Norman Osborn about the project being the creation of his predecessor, the background behind Norman changes from a wall with a railing above it, to him standing in front of the test chamber, between shots.
The right-handed Peter Parker always used an average right-handed camera, but in one take during the balcony scene, we see a reversed image of the scene and it appears that he is holding a left-handed camera.
During the World Unity Fair, Mary Jane is trapped on a piece of balcony that falls away, quite clearly hanging off and separated from the rest of the balcony. However, after it cuts to Spider-man and back to her, the piece of balcony is attached and falls away a second time.
The windows beneath the balcony at the World Unity Festival are destroyed by the Green Goblin's pumpkin bombs, but they are only on fire and not shattered when Spider-Man dives off the balcony to rescue Mary Jane.
When Peter Parker looks up at the balcony where Mary Jane and Harry Osborn are standing during the World Unity Festival, he looks directly up from his position on the ground. However, when Harry looks back down on the ground to see Peter Parker, he is looking towards his far left side.
On the morning Peter does a little web practice, he talks to his Aunt at the door. After closing the door, we see that there are webs all over the room, even in the path he just walked to get to the door.
When Peter spills the tray of food on Flash and runs out of the cafeteria, Flash has food on his shirt. Once in the hallway, the food disappears, reappears (but it's a smaller amount), and then disappears altogether.
During the alleyway fight involving Mary Jane, Spider-man yanks all four men off of her with webbing. The webbing is strong enough to drag them across the street, however when he hops down off the lamppost, all the webbing is gone.
When Peter smacks the food tray onto Flash's back, the tray hits Flash on his left side. The stain from the food is shown on this side in differing sizes except for the moment Flash stands up and turns around, saying "Parker" - in which it is shown to be a huge stain on his right side.
During the World Unity Festival, when Peter Parker sees a huge piece of stone from the building falling rapidly, the camera shows a man and woman facing the camera and cowering. In the next shot in which Peter uses his web slingers to pull the people to safety, they are shown standing up, looking straight up, with their backs to the camera.
During the field trip, Harry, Peter, and Mr. Osborn are talking outside the building, the students are walking up the stairs in the background during one shot and not in the background at all in the next. Later they return in the background once again.
During the World Unity Festival, when Peter runs toward camera and unbuttons his shirt, the Spider-Man suit only extends to the top of his chest. However, when he removes his mask during the Thanksgiving sequence, the costume clearly goes all the way to his neck.
The morning after the spider bite, Peter comes downstairs and
startles his uncle. Just before he does that, Uncle Ben can be seen holding a cup with both of his hands, yet in the next shot, he is holding it with just one hand. He also pulls the cup away from his face "twice", once in each shot.
At the World Unity Festival, a block of stone falls from the balcony and is headed for a man and his wife. When they are shown flinching at the stone about to hit them, the man is on the left side of his wife. When Peter spins his web onto them to save them, he is on the right.
After the kiss, MJ tries to adjust the mask of Spider-Man, by pulling it up, of course. But when the mask reaches the end of his mouth, Spider-Man launches his web to leave and the mask is magically adjusted.
In the World Unity Festival, Peter can be seen looking up at Harry and Mary Jane from the ground. Harry then looks back at Peter clearly recognizing him. Later when Mary Jane falls from the balcony, the balcony is much much higher than earlier when Harry looked down at Peter.
When Peter walks behind MJ on the street, the school bus turns the corner and approaches slowly in the background. Ten seconds later, when the bus is shown a third time, however, it is still passing by the same vehicle.
When Peter is first becoming aware of his "spider sense" in school as Flash is approaching from behind, he sees a fly and a paper airplane moving in very slow motion. The fly has two wings when in reality, flies have two sets of wings, the main wings plus a smaller "halter" wings underneath (to maintain balance in flight), for a total of four wings.
When Peter is walking behind Mary Jane, on the way to the bus, he stops and talks to himself. over his left shoulder is a car parked, with a Massachusetts license plate. Cars from many different states visit or pass through New York on a daily basis.
When Peter is practicing his web-slinging in his room and catches the lamp to pull it towards him, it doesn't appear to move very fast off the chest and looks more likely to fall on the floor before the shot changes. When it cuts to Peter, it speeds past his shoulder a lot faster to smash on the wall behind him. Furthermore, Aunt May would have easily seen the amount of webbing all over the room through the gap in the door as she's talking to him.
When Spider-man swings MJ to safety and we see them swinging close-up, not only is MJ's hair blowing in the wrong direction, but Spider-man's head, neck and shoulders appear to be stiff as if it were a mannequin she is holding on to for the sake of the shot.
When Spider-Man is chasing the carjacker, a brief shot from the inside of a police car shows they are moving at only 15mph. This is not nearly as fast as the obviously sped-up sequence leads us to believe.
Although Peter is supposed to have perfect vision after his bite, Tobey Maguire's contact lenses are visible at various points throughout the movie. It could be that Peter took to wearing "blank" contact lenses to account for the fact that he no longer needs glasses, but is more likely to be a true goof.
When Flash tries to beat up Peter, a crowd of students gather around them. When Peter knocks Flash down the hallway, though, those students have created an alley for Flash to fly through. Given the superhuman speed with which Peter punched Flash, none of the students would have had time to step aside, anyway - they would simply have been bowled down.
When MJ is in front of the spider cage, just prior to Peter being bitten, a spider on a thread can be seen just over Peter's right shoulder in the medium shots, and directly underneath the spot where he will be bitten. It disappears in the final cut before closeup coverage begins. Notice also that the focus changes on the background, indicating the use of different lenses or different spikes for the shots used.
After depositing MJ on the green lawn at the church, Spider-man obviously jumps off of a mini trampoline over the hedge. As MJ runs behind to follow, she clearly has to step around the mini trampoline to approach the hedge.
When Spider-man jumps on the car, there's an in-car shot of the carjacker firing up at the roof. When the camera goes back to Spider-man on the roof of the car, the bullet hole switch position. In fact, if the bullet holes had stayed in position, Spider-man would have been killed.
When we see an exterior view of the Oscorp office building, just before Norman Osborn's meeting with the board, the main sign is for Oscorp. However, at each corner of the building, the ING logo with its distinctive lion can clearly be seen.
During the World Unity Fair, Times Square alternates between the real Times Square and the digitally altered one created for the movie. The main difference is that the altered version doesn't have the gigantic Cup-O-Noodle sign.
The Cup-O-Noodle sign can be clearly seen twice, and the bottom of it a 3rd time. First time when you first seen the Goblin flying in the distance, and the 2nd is during the shot from behind in when she flies past the Earth balloon before throwing the first bomb, the sign is on the left. Then when Goblin is flying off, you see the bottom of it.
The extras at Columbia University are wearing cold weather clothing, appropriate for the seasonal setting. Students in the background wearing shorts and t-shirts blow the illusion and reveal that the scene was shot on a warm day.
When Mary-Jane is attacked in the rain in the alley Peter is shown running along the roof changing into his costume, you can clearly see he is already wearing his mask, but when he fights the thugs he has no mask.
When the car-jacker walks into the deserted warehouse, he has the gun in his left hand and the money bag under his right (flipped image). Next cut is to him reloading, and the gun is back into his right hand.
It is established that Peter is right-handed, but when he is designing his Spider-Man costume, there are two quick shots of him drawing with his left hand. These shots are most likely flipped images, rather than a suggestion that he's ambidextrous.
The goof items below may give away important plot points.
When Spider-Man is hit by the bomb at the end in the destroyed hospital, the Green Goblin comes down and rises his eye lids on his mask, "misery, misery, misery, that's all that you've chosen," Then in the next shot where he says "I offered you friendship and you spat in my face." The eye lids are closed, but when he is about to stab Spider-Man with the trident, the eye lids are opened, and when he is pushed onto the brick wall, they are closed, they stay closed the rest of the scene.
When Spider-man's arm is cut during the fight with Green Goblin in the burning building the cut runs diagonally down his arm toward his thumb. In the Thanksgiving dinner scene when Norman Osborn sees the wound it runs diagonally down his arm toward his little finger.
When Spider-Man jumps over the oncoming bladed glider, which hits the Goblin instead, the glider seems to hit at about stomach level and pin the Goblin to the wall. But in the next shot, it is lower, seeming to have hit at groin level, to allow the Goblin to slump forward across the glider.
When Spider-Man fights the carjacker, they enter the building on the ground floor but then the carjacker trips and falls out what appears to be a 3rd story window even though neither of the two characters can be seen climbing to a higher level during any part of the fight sequence.
When Peter Parker gives chase to the killer of Uncle Ben, he wears the same outfit he wore when fighting Bonesaw. Peter climbs a building and then jumps from building to building. He winds up on a dome shaped roof and works up the nerve to web sling. The figure shooting the web from that dome is Peter in his Spider-man suit which does not invent until later in the movie.
In the bridge scene, after the Green Goblin destroys one of the stations, he flies down, grabs the wire for the cable car it and pulls it upwards with him to the top of the bridge. However, despite that he's strong enough to hold it himself, the glider he's on barely has enough thrust to carry a humans weight on it, so the chances of it being able to fly upwards whilst holding onto a wire attached to a huge cable car is unlikely.
When Peter Parker calls MJ on the phone and gets her answering machine, the Green Goblin answers and he taunts Peter calling him Spider-man. This is all being recorded by the answering machine, so MJ might listen to it and discover that Peter is Spider-man. But not if Goblin erased the message immediately after the call, which is what most likely happened.
When Peter realizes that Uncle Ben's killer is the thief he let go at the wrestling arena, his flashbacks are very inconsistent to what really happened. The gunman originally nodded his head very quickly and said thanks sounding out of breath. In Peter's flashback, the gunman stands there and smiles and says thanks as the elevator doors close. This is to show the subjective memory of a grieving Peter in contrast to the objective portrayal of the event.
In the final conflict, Spider-man tries to save both the falling children in the transportation device and Mary Jane all the children and the teachers are screaming. However, one man inside with the children who either controls the device or is a fellow teacher seems somewhat relaxed as it is falling. It is unlike that he would be so relaxed as it is falling while everyone is going crazy.
Right before the Thanksgiving dinner, Spider-Man and the Goblin get in a fight in a burning building. In the outside shots of the burning building, the green leaves on the trees clearly indicate that it is summer time in New York. After the Thanksgiving dinner, Aunt May is attacked by the Goblin. In the outside shots of her home, once again all the trees are green.