A year after the Devil's Breath incident, Miles Morales (Nadji Jeter) is now being mentored by Peter Parker (Yuri Lowenthal) and plans to help keep New York safe while honoring the memory of his late father Jefferson Davis (Russell Richardson). After they stop a disastrous prison transfer caused by Rhino (Fred Tatasciore), Peter leaves Miles to take care of New York City for him, while on vacation in Europe in order to help his girlfriend Mary Jane Watson with her news coverage on Symkaria as her photographer.

Miles goes back home to celebrate Christmas with his mother Rio (Jaqueline Pinol) and best friends Ganke Lee (Griffin Puatu) and Phin Mason (Jasmin Savoy Brown). The next day, Miles attends his mother’s campaign rally, but it is interrupted by The Underground, a tech inspired terrorist group that has a vendetta with the Roxxon Energy Corporation.

During the crisis, Miles learns that Phin is the leader of the Underground operating under the alias "The Tinkerer" and her motive is to avenge the death of her late brother Rick (Todd Williams) who suffered from a chemical poisoning while working on Roxxon's new energy source Nuform. He later learns that she plans to destroy the company by blowing up a newly built Roxxon Energy plaza with an overloaded canister of Nuform to prove the energy source's disastrous side-effects that Roxxon CEO Simon Krieger (Troy Baker) had been covering up.

Miles runs into a series of setbacks which include an internal conflict with his overprotective uncle Aaron Davis (Ike Amadi) who operates as Prowler; failed attempts to reason with Phin (Who learns about his identity as Spider-Man), fighting a technologically advanced Rhino now working for Roxxon, and his mother learning the truth about his Spider-Man activities who then supports him. At the climax of the crisis, Aaron helps Rio and the neighbors evacuate Harlem when both The Underground and Roxxon start fighting on the streets of Harlem while Miles confronts and defeats Phin when the overloaded Nuform generator gives off a series of destructive shockwaves.

Miles attempts to absorb Nuform's energy to negate the blast, but is unable to contain it. Seeing that the Nuform energy is going to kill Miles, Phin absorbs the energy from him and dies in an explosion. Miles's identity as Spider-Man is revealed to the people of Harlem who promise to keep it a secret. Four weeks later, Krieger is arrested for his crimes after Aaron testifies against him in exchange for for a reduced sentence.

Roxxon has also been hit with a series of lawsuits. Miles resumes his duties as Spider-Man with Peter who has returned from Symkaria. In a mid-credits scene, former mayor Norman Osborn (Mark Rolston) demands his terminally ill son Harry's release from his stasis, despite warnings from a reluctant Dr. Curt Connors that it is too dangerous to do so. In a post-credits scene, Miles leaves a science award cube at the top of the church in memory of Phin.