After Michael is impaled, Selene walks into Alexander's office with blue eyes. When she is walking up the stairs, there is a far shot of her, and her eyes are brown. When the camera shows her up close again, her eyes are blue. However, the vampires' eye-colors change throughout the movies. They are often brown (natural eye-colors of the actors/actresses), but change to blue when the intensity of a situation increases. For example, Selene's eyes change from brown to blue during her fight/searching sequences in both movies, as well as her sex-scene in this film. Kraven's eyes exhibit the same behavior in this film and in the first one also.
When Michael is at the pub and is being chased by the police, he jumps onto a police car and leaves a rather large dent. A moment later when he runs into the woods, there is a shot of the car and the dent is gone.
During the fight on the truck, the vehicle is severely damaged, most notably on the passenger side when Marcus is crushed between it and the cliff face. Upon arriving at the warehouse, however, there is almost no damage whatsoever.
At the end of the film, sunlight is shining straight down through a hole in the cavern ceiling, indicating that the sun is directly overhead (ie, it is noon). But in an exterior shot, it is dawn and the sun has barely cleared the horizon.
When Alexander's strike team jump from the helicopter into the river near the end they are wearing breathing masks of the style firefighters use, but when they hit he water they have scuba masks, then after they swim underwater and surface they have the original style on.
When Selene is firing her pistols during the final action sequence, she reloads multiple times with clips from behind her. In preceding and following shots she has nothing on her back that could contain one or multiple clips for her pistols.
When Michael tears the paint cans to throw them at the windows, so that sunlight would not enter and damage Selene, you can clearly see his hands full of paint, but in the next shot, his hands are clean.
After escaping Marcus right before dawn, Selene is burned by the rising sun. She and Michael are forced to take shelter in a warehouse and Michael rips a first aid kit shaped like a bathroom medicine cabinet off the wall. When he sets it down next to Selene, the kit/cabinet has changed into a tackle box.
When Selene first enters Tanis' compound and falls into the tunnel she is attacked by a Lycan and draws a knife. She drops the knife which falls to the ground and is shown with some dirt across the blade. When she reaches back for it to kill the Lycan the blade is clean and facing the other way.
The signs at the gate of the monastery where Andreas Tanis is hiding are visible (at around 42 mins) and intact. When passing the gate (at around 43 mins) the sign saying "Hazorzo kutya" is visible in the background and partially broken.
(at around 10 mins) The coordinates shown for the "safe house" (Location 254, Long 32 12 23, Lat 60 21 44) link to a location in Iran (near to the Afghanistan border) while the movie takes place in Hungary (recognizable from signs as the one saying "Hazorzo kutya" when entering Tanis' hiding place).
During several scenes in the fortress, Selene empties her guns. After her pistols are empty the slides lock back but you can still hear click, click, click. That would be impossible as when the slide locks back you can't pull the trigger. The same happens when she empties her rifle and the bolt locks back.
Marcus brings down a helicopter by pulling on the attached cable. This is implausible and physically impossible. Regardless of one's sheer strength, unless he is anchored to the ground, the helicopter would have lifted him rather than get pulled down.
The signs on Tanis' gate do not translate well. The first sign (Freszolitas Hazorzo Kutya) is two different languages. Using Google Translator, I discovered that "Freszolitas" is a word in Esperanto (whatever country that is) and doesn't translate into any word in any other language, while "Hazorzo Kutya" translates to "Watch Dog". In order for the first sign to match what the English sign states, it should read "Figyelmeztetés: Orzokutya" which is Hungarian for "Warning: Guard Dog".
During the pub scene the newscaster woman and the two soldiers speaks Hungarian in a foreign accent - not to mention the grammatical errors and mis-spelled words - revealing that they are not Hungarians but foreigners who can speak the language. When Michael enters the pub the background newscaster speaks the correct language.
In the scene where Marcus is entering the area after the movable wall unlocked with the key, where William's prison is, you can see a hole in the ceiling where light is entering the room. There would no purpose for a unmovable wall if you could enter the room through a hole in the ceiling.
The goof items below may give away important plot points.
After Selene finishes off Markus by throwing him into the helicopter's rotor blades they slow down to a halt; one is broken, two are partially broken or bent, one is intact (visible in the background at 01:31:40). In the next shot they are all broken (visible in the background at 01:33:40).
When the 'gas grenade' is used for the first time in the safe house, it pops open, sprays the gas, then the two electrical rods pop out and spark causing the explosion. But when Alexander Corvinus uses the grenade to kill himself later in the film, the rods pop up before the gas is released, then they spark to cause the explosion. Apparently, the two grenades are of slightly different models, and work slightly different.