Peter has begun to feel secure in his life and plans to propose to Mary Jane. One night in a park, while Peter and Mary Jane are on a date, a small meteorite crashes nearby, and an alien symbiote oozes out, attaching itself to his moped. Meanwhile, escaped convict Flint Marko falls into a particle accelerator which transforms him into a shape shifting sand monster. Peter's best friend, Harry Osborn, who seeks vengeance for his father's death, which he believes Peter caused, attacks him. The battle leaves him with short-term amnesia, making him forget his vendetta.

Later, during a festival honoring Spider-Man for saving Gwen Stacy’s life, Marko attempts to rob an armored car, and overpowers Spider-Man. Captain Stacy later informs Peter and Aunt May that Marko is the one who killed Ben Parker, and a vengeful Peter waits for Marko to strike again. The symbiote bonds with his costume while he is asleep, and Peter discovers that not only has his costume changed, but his powers have been enhanced as well. The black suit also alters Peter's personality, making him more violent, exemplified by a near lethal attack on Marko during a battle underground.

Affected by the suit, Peter exposes and humiliates Eddie Brock Jr., a rival photographer at the Daily Bugle, who sells fake pictures of Spider-Man. The shift in Peter's personality alienates Mary Jane, whose stage career is floundering, and she finds solace in Harry. Harry then recovers from his amnesia and threatens to kill Peter unless MJ breaks up with him. After Mary Jane leaves Peter, stating she is in love with another man, Harry meets him at a restaurant and claims to be the other man. Harry speeds off before Peter can confront him, but Peter finds him at the Osborn mansion. With the help of the black suit, Peter is victorious in a brutal fight which leaves Harry's face disfigured.

In an effort to make MJ jealous, Peter brings Gwen to the nightclub where Mary Jane works and gets into a fight. In the scuffle, he accidentally throws MJ to the floor. Peter realizes the symbiote-suit is changing him for the worse. He runs out of the nightclub and goes to a church bell tower to be rid of it. Initially he is unable to pull the suit off, but the sound waves from the church bells weaken the symbiote, freeing Peter. Eddie Brock is at the same church, praying for Peter’s death, and the symbiote falls from the tower and takes over his body. The newly powered Eddie finds Marko and suggests that they join forces to destroy Spider-Man.

The pair use Mary Jane as bait to lure Spider-Man to confront them. Peter approaches Harry for help, but is turned down and leaves. However, Harry learns the truth about his father's death and arrives in time to rescue Peter, teaming up against Brock and Marko. As the fight progresses, Brock attempts to impale Peter with the glider, but Harry sacrifices himself and is fatally wounded. Peter recalls how the church bell's toll weakened the symbiote, and frees Eddie from it by clanging several pipes together. Peter throws a pumpkin bomb at the symbiote just as Eddie, who has grown attached to its power, attempts to re-bond with it; Eddie and the symbiote are destroyed.

After the battle, Marko tells Peter that he had no intention of killing Ben Parker, and that it was an accident born out of a desperate attempt to save his daughter's life. Peter forgives Flint, who dissipates and floats away. Peter and Harry forgive each other as Harry dies with Mary Jane and Peter by his side. Afterward, Peter and Mary Jane begin to mend their relationship.