Continuity mistake: Inside the school lab, the Lizard walks to Peter on the floor and a stool next to him suddenly appears for him to pick up. (01:35:00)
Continuity mistake: In the scene where the lizard and Spiderman are fighting in the sewers, the lizard scratches Spider-Man's chest, causing tears down the front of his suit and chest. He actually scratches straight down his chest, but from then on, whenever Spiderman's chest is shown the scratches are diagonally across him.
Continuity mistake: In the scene where Peter Parker is fighting on a train he tells the last man standing 'not my board', the last man is black but after hitting Parker and the board breaking the next shot is one of the guys he has already knocked down who is white fighting him after the board breaks.
Continuity mistake: When Gwen visits Peter at the end of the movie, you can see that the glass door with the house number "36" painted on it has been replaced from when he broke it earlier in the movie, and it looks brand new. Once she leaves and he turns to Aunt May, the numbers have returned to their earlier weathered and peeling state.
Factual error: There is a scene in midtown Manhattan toward the middle of the film outside a NYC subway station, which according to the sign on the subway entrance is at 6th Avenue and 39th Street. There is no such subway station on those streets.
Continuity mistake: During the scene where Uncle Ben is talking to Peter in Peter's bedroom, on his desk there is a solved Rubik's cube. Uncle Ben picks it up, and twists it slightly. It then suddenly appears on the opposite side of the desk next to Peter, too far away for Ben to have placed it there, fully solved again.
Continuity mistake: When Peter asks Flash to stop bullying the other student in the outdoor eating area, right before Flash punches Peter, Peter is carrying his camera on a strap on his right shoulder. As the shot changes to Flash punching Peter, the camera strap disappears before Peter even gets hit and falls to the ground. Later we find Peter's camera smashed on the ground next to his backpack and skateboard.