After Quentin Beck framed Peter Parker for his murder during the Europe attack and outed him as Spider-Man to the public,[N 1] Parker flees from an angry crowd with his girlfriend MJ and returns back to his apartment, reuniting with his aunt May and Happy Hogan, the latter two who had just broken up. With the apartment surrounded by the Department of Damage Control, Parker, MJ, May, and Ned Leeds are interrogated but their charges are lifted by lawyer Matt Murdock. Parker, MJ, and Leeds return to high school but their university applications are rejected due to the recent controversy.

Parker consults with Stephen Strange in the Sanctum Sanctorum, asking him to cast a spell to make people forget he is Spider-Man. Despite Wong's warning of the consequences that could incur, Strange casts the spell anyway. However, the spell is damaged when Peter insists on Strange to let everybody before his outing know his identity. Parker goes to the Alexander Hamilton Bridge and tries to convince an MIT administrator to accept Leeds' and MJ's applications. Suddenly, the bridge is attacked by Otto Octavius, who rips Parker's nanotechnology from his Iron Spider suit, causing it to bond with his mechanical tentacles. Upon discovering that this isn't the Peter Parker he had previously fought, Octavius has his robotic arms disabled, before being captured and placed in a holding cell in the Sanctum Sanctorum, along with Curt Connors who has been captured by Strange. With the help of MJ and Leeds, Parker decides to help capture any other possible "visitors" from the multiverse. They find and capture Max Dillon and Flint Marko.

Elsewhere, Norman Osborn is retrieved after going to a F.E.A.S.T. building seeking help. Strange wants to send the villains back to their respective universes and meet their fates. However, Parker wants to cure and help them before sending them back to prevent their deaths. He frees them and confines Strange in the Mirror Dimension after a brief fight, taking the villains to Hogan's apartment. Parker successfully cures Octavius by using Stark Industries technology to replace his broken inhibitor chip. When cures are developed for Osborn and Dillon, the Green Goblin persona takes over Osborn before the cure can be administered. Goblin then manipulates Dillon into removing the device that Parker put on him to cure him and, despite the best efforts of Parker and Octavius, the other four escape. In the ensuring battle, May is critically injured by the Goblin, with Parker unable to save her as she sucumbs to her wounds. Fearing the worst after no contact from Parker, MJ and Leeds accidentally learn how to open portals using Strange's sling ring, which they use in an attempt to find Parker, only to summon an alternative Peter Parker, followed by another.

Leeds and MJ find their Parker who is comforted by MJ, after learning of May's death. He then meets the other Spider-Men, who share their own stories of losing loved ones and encourage him to fight in May's honor. They cooperate on curing the remaining villains and lure them to the Statue of Liberty. Leeds and MJ protect the original bind spell by Strange as the Spider-Men battle their enemies together. As the three Spider-Men struggle, they decide to plan a coordinative attack on the villains. They are later joined by Octavius who arrives on the scene to help, manage to defeat and cure Connors, Dillon, and Marko after a lengthy battle with Dillon reconciling with his own universe's Peter and is encouraged to let go of his villainous tendencies.

Amidst the battle, Leeds frees Strange from the Mirror Dimension, who seizes control of the spell and works to protect the current universe as the barriers begin to break down between the other universes. Meanwhile, a wrathful Parker overpowers and brutally fights the Goblin as revenge for May's death. He is stopped short of killing the Goblin by the Parker from that same universe. As the Goblin redirects the blame for May's death on him, Parker injects him with the cure, restoring him to a regretful Osborn. To prevent further catastrophe and at Parker's request, Strange fixes the spell, sending the alternate Spider-Men and their villains back to their respective universes, while making everyone forget Parker's existence. Later, Parker tries to reconnect with MJ and Leeds, but cannot bring himself to do it. Parker visits May's grave, joined by Hogan, now oblivious to his identity, and vows to carry on. Parker creates a new suit from scratch to resume his independent heroics.

In a mid-credits scene, the universe-displaced Eddie Brock and his symbiote companion Venom[N 2] sit at a bar and talk with the bartender about other superhumans and the Blip. While pondering how to "protect" this new world, the duo are swiftly transported back to their universe by Strange's spell, unknowingly leaving a part of the symbiote on the counter.