Months after the incident of the movie, Dr. Curt Connors' incarceration, Peter Parker and Gwen Stacy sneak into the restricted areas of Oscorp after hours to investigate rumors of the company continuing Connors' cross-species experiments. They are caught by new Oscorp director Alistair Smythe, who confirms the rumors and gives them a tour of Oscorp's restricted areas to see the experiments themselves. However, the cross-species react to Peter, a cross-species himself, and break out, infecting numerous scientists, including Smythe and Gwen, with a deadly virus they are carrying. A giant scorpion bites and infects Dr. Smythe and a giant vermin rat bites and infects Gwen on her left shoulder. Donning his Spider-Man suit, Peter gets the infected to quarantine, but fails to stop the cross-species from escaping into the city.
After a fight with S-01, a massive cross-species hunting robot created by Smythe, Spider-Man reluctantly breaks Connors out of the Beloit Psychiatric Hospital to help find a cure for the virus. Spider-Man and Connors set up a laboratory at the apartment of Aunt May's friend Stan, and begin development of an antidote while keeping in close contact with Gwen and Smythe via webcam, unaware that Smythe is developing his own "cure" using nanobots that destroy the host from the inside out. After defeating Rhino, one of the escaped cross-species, Spider-Man retrieves Connors' research from a secret Oscorp lab before it gets destroyed. While there, he meets Whitney Chang, an undercover investigative reporter, who enlists Spider-Man's help in unveiling Oscorp's suspicious activities. Spider-Man also collects a blood sample from the rat cross-species, Vermin, for Connors' antidote, while battling a group of "Hunters" - a new type of cross-species hunting robot who identify him as one.
Spider-Man eventually delivers Connors' antidote to Gwen at Oscorp, now under complete quarantine. However, a skeptical Smythe takes and tests it on himself, claiming that, if it works, Oscorp will replicate it and he will take all the credit as the city's savior. However, instead of curing him, the antidote causes Smythe to lose the use of his legs and sanity. Enraged, he commands his robots to eliminate Spider-Man, who escapes after destroying S-02. Realizing that Spider-Man is a unique cross-species who maintains his humanity, Connors creates a new antidote using his DNA, which Spider-Man again delivers to Oscorp, fighting his way past security and the Scorpion cross-species. The antidote works, saving Gwen's and the other scientists' lives.
As the CDC quarantines the city due to the advancement of the virus, Spider-Man, while waiting for Connors to replicate the antidote, returns to his heroic activities. He foils a bank robbery orchestrated by Felicia Hardy, who escaped from Beloit during Spider-Man's initial attempt to break Connors out; assists in the re-capture of Scorpion; stops Rhino from poisoning the city's water supply, and rescues the district attorney from the Iguana cross-species. Meanwhile, Smythe, having been fired from Oscorp for his actions, discovers Spider-Man's secret identity and kidnaps Connors from their apartment, challenging Spider-Man to come and rescue him at Oscorp's robotics facility. While he rescues Connors, Spider-Man is captured by Smythe, who injects him with a nanobot serum that strips away his powers, and reveals his plan to spread the same serum across the city using his greatest invention yet, S-03, unaware that the nanobots are slowly killing the host. Spider-Man escapes and makes his way across a Manhattan consumed by chaos to reach Connors' secret lab, where he reunites with him and Gwen.
While Connors transforms into the Lizard and leaves to try and stop Smythe, Gwen resuscitates the dying Spider-Man with an AED. Realizing that electricity can destroy the nanobots, Spider-Man leaves to assist Connors and boards S-03 with Whitney Chang's help, whereupon he electrocutes himself to destroy the nanobots, regaining his powers. Spider-Man and Connors defeat Smythe, but Connors then succumbs to the Lizard's consciousness and flees into the sewers. Leaving a remorseful Smythe, who regained his sanity, for the police, Spider-Man pursues the Lizard, along the way defeating Natie, the piranha cross-species, and saving Gwen, who has reprogrammed a Hunter to administer the cure across the city. Eventually, Spider-Man defeats and cures the Lizard, whereupon Connors willingly returns to Beloit. As New York slowly returns to normal, both Spider-Man and the Lizard are credited as the city's saviors. Peter and Gwen learn from the news that Smythe has escaped custody but decide to deal with it later.
In a post-credits scene, Smythe, having regained the use of his legs due to being in the final stages of his infection, returns to his lab and commands one of his Hunters to kill Spider-Man, choosing to die rather than become a cross-species or take Peter and Connors' cure.
