Continuity mistake: In the scene where Peter is riding his scooter to Mary Jane's play, after the music fades out you see a black convertible being chased by several police cars. The black convertible is rammed forcibly, and you see many hideous dents on the side of the car. Thirty seconds later, after Peter is done talking to the kids, we see the same car from the same side it got rammed into, but the hideous dents have vanished.
Plot hole: At the end, when Peter saves Mary Jane from the collapsing structure, he jumps up in the air and swings her to safety. However if you look at the web he is holding onto it is hanging down vertically. What did he attach the web to that was directly above him, the sky? Every bit of the structure around him was/had collapsed and the only thing remotely close to swing from was the cranes. Yet we see that was quite a distance away, so Peter would have had to shoot the web away from himself not above him.
Continuity mistake: As Peter stops the elevated train with many webs, a tear appears in his suit on the inside left arm during a closeup. Later, when his arms are still outstreched, there are many shots where you can see that the tear has disappeared; for example, a few shots later when he is still trying to stop the train; when the passengers are lifting him over their heads into the train; and many others.
Continuity mistake: On his last pizza delivery before he gets fired, when it first cuts to the receptionist and the room that she is in, the clock is very high up in this shot, above the receptionist's head, then in the following shot when the receptionist is looking in Peter's direction, the clock is much lower.
Trivia: Spider-Man creator Stan Lee makes his standard cameo appearance for this film when Spider-Man and Doc Ock are fighting on the side of the building. The first shot of the street during the fight shows a woman being pushed out of the way as some debris falls; the man pushing her is Lee.