In an alternate 1899, several high profile crimes occur in various nations throughout Europe, each allegedly carried out by nationals of other countries, leading Europe to the brink of war. An emissary of the British government, Sanderson Reed (Tom Goodman-Hill), arrives in a gentlemens club in Kenya, hoping to recruit the legendary, but now aged, hunter and adventurer Allan Quatermain(Sean Connery) to investigate the situation. However, Reed finds that Quatermain's sense of patriotism has declined over the years, and he refuses to offer his services. Suddenly, a group of armoured men armed with machine guns appear, and begin killing the club members. Quatermain defeats the attackers single-handed, giving chase to finish the last one, thereby surviving the club's destruction by a bomb planted by the men. Quatermain reluctantly agrees to return to England, deciding that a full scale war between the nations of Europe will quickly spread to Africa.
Some time later, Quatermain arrives in London and meets the mysterious 'M'(Richard Roxburgh), who explains his plan to assemble a League of Extraordinary Gentlemen to combat the threat of The Fantom, who is the true mastermind of the current crisis, and ensure world peace, by stopping him from destroying Venice. In short order, Quartermain meets Captain Nemo(Naseeruddin Shah), the invisible gentleman thief Rodney Skinner(Tony Curran), and Mrs. Mina Harker(Peta Wilson), whom 'M' introduces as a well-regarded chemical scientist. Skinner had stolen an invisibility formula from its creator and is now helping the government in return for an antidote.
'M' sends the group to meet up with the mysterious Dorian Gray(Stuart Townsend), whom they hope to convince to be another member of the League, but he refuses to join. While talking to Gray, Quatermain points out that a painting is obviously missing from a wall. Suddenly, the 'Fantom' and his men arrive, and the whole room erupts in a blazing gunfight, but one henchman reveals himself to be a friend of the group. During the fight, Dorian Gray's physical invulnerability is revealed, and The Fantom escapes. The fight ends with Mina being held with a knife at her throat. Mina's eyes suddenly turn red and she rips the throat from her assailant and feeds voraciously from his neck. Mina then explains her background, regarding her encounter with Dracula and her resultant powers. It is revealed the friendly 'henchman' is an American, Secret Service Agent Tom Sawyer(Shane West), whom a mildly impressed Quartermain allows to join them. The previously reluctant Dorian changes his mind and agrees to join the League. The League then takes Nemo's submarine, the Nautilus, to France to round up the Leagues last recruit.
In Paris, Quartermain and Sawyer capture Mr. Hyde(Jason Flemyng) who quickly reverts to the meek and haunted Dr. Jekyll. He is happy to help when he learns that the Queen will allow his return to London if he does so. With the team complete, the Nautilus sets off for Venice. The group worry there is a traitor in their midst when flash powder is found in the wheel room of the Nautilus, and a vial of Jekylls serum is determined to be missing. Naturally, all think that the invisible thief, Skinner, is the culprit, but nothing can be done about it since Skinner is nowhere to be found.
The Nautilus sails up the narrow canals of Venice, stopping under a bridge where it can go no further. A series of bombs has been planted under the city; they start to detonate and begin toppling buildings in a domino effect, one explosion after another. The team decides that knocking one of the buildings out of the sequence is the only way to stop the chain of explosions. Nemo has a missile that can be fired from the Nautilus at the building in question, but only if a beacon can be set in place. Since Nemo can track his "automobile," allowing it to serve as the beacon, the League piles in, with Sawyer at the wheel, and go racing along the streets of Venice.
Along the way, Gray and Mina jump out separately to fight groups of henchmen. Quatermain also jumps from the vehicle to give chase to the 'Fantom' on foot. The two fight in a graveyard, where the 'Fantom' loses his mask. Before he makes his escape, the 'Fantom' is revealed to be none other than the mysterious 'M'. At the same time, Sawyer crashes the car into the target building, while firing a flare, which signals Nemo to launch his missile. The building is destroyed, the chain of explosions stops, and Venice is saved. Meanwhile, back at the Nautilus, the first mate Ishmael is met by Gray and blames Skinner for their troubles, but is proven wrong when Gray shoots him.
The League regroups at the Nautilus, where Quatermain reveals that "M" is behind the whole thing. The dying Ishmael appears, denouncing Gray as the traitor. A small submersible vessel suddenly breaks from the Nautilus: it is Gray, making his escape with a smile and a wave to the assembled team. Nemo sets the Nautilus in pursuit, but along the way, a record is found and played on a gramophone. The voice of 'M' floods the room, as he reveals the scope of his plans. Everything leading up to this moment was a ruse so that 'M' could steal physical elements from each of the League members, so as to construct an army of super-powered soldiers; Captain Nemo's science, Jekyll's formula, Mina's blood, and a sample of Skinner's invisible skin. He also explains that he needed Quatermain only to capture Mr. Hyde. 'M' seeks to profit by starting a world war and selling armaments and weapons based on the powers of the League to the combatant countries. He blackmailed Gray into helping him by stealing his enchanted portrait, the missing picture from his home. As the record is played, it's second unheard signal sets off three bombs in the ship, but Mr. Hyde is able to stop the ship from sinking, earning the congratulations of Dr. Jekyll. Following a signal from Skinner, the Nautilus follows to the Asiatic Arctic and the League travel across the frozen wastes to a cave overlooking an industrial fortress. Skinner approaches (he had fled when he learned of their suspicions of him), and tells them that 'M' has a number of scientists and their families held as hostages and slaves in his munitions factory. The workers are constructing Nautiloi (copies of the Nautilus), while the scientists are working on an army of Hyde-like brutes, invisible spies, and vampire assassins.
Splitting up, the League infiltrates the factory. Nemo and Hyde attempt to free the scientists and their families, while Sawyer and Quatermain go after 'M'. Mina goes in search of Gray, while Skinner sets off to plant some explosives. Nemo and Hyde run into the second-in-command, Dante, who drinks a very large dose of Jekyll's formula and transforms into a huge, hulking monster to combat Hyde. Mina has her showdown with Gray, which leads nowhere (as they are both immortal) until she confronts him with his enchanted portrait, causing him to decay before her eyes. Quatermain confronts 'M' in his lair and reveals his deduction that 'M' is none other than the supposedly dead Professor James Moriarty (nemesis of Sherlock Holmes). As Hyde is battling Dante, the explosives go off and Dante is crushed to death by falling debris. Quatermain, while holding Moriarty at gunpoint, sees in a reflection that an invisible man (Sanderson Reed), has Sawyer held at knife point behind him. He turns and shoots the invisible villain, only to be stabbed in the back by Moriarty, who makes another escape, fleeing across the ice. Quatermain is mortally wounded; Sawyer is forced to use the marksmanship skills that Quatermain had taught him. He shoots Moriarty before he can leave in his stolen submersible vessel. Moriarty's case, containing the secrets that Gray had stolen from the League, falls into the ocean, and Moriarty dies on the spot. Quartermain also dies soon after.
The group assembles in Africa to bury Quatermain. As the group sadly departs, a tribal witch doctor takes handfuls of dirt from Quatermain's grave and begins a ritual chant. We are reminded of a witch doctor's pronouncement, recounted by Quatermain at the beginning of the movie, that "Africa would not let [Quatermain] die." The earth shakes violently, making the rifle that Sawyer had left on the grave shake. Lightning strikes Quatermain's grave right before the screen cuts to black.