Nick Fury has been infected by nanite versions of Doctor Octopus' Octo-Bots. While the others face off against Doctor Octopus, Spider-Man and Power Man work with Ant-Man to enter Nick Fury's body and get the Octo-Bots out of him.

Peter Parker learns Doctor Octopus has teamed up with Arnim Zola and Hydra to attack the Tri-Carrier, leaving S.H.I.E.L.D. caught off-guard and Spider-Man left to single-handedly stop the assault. He assembles a new team, including an apprehensive Scarlet Spider, in order to stop the villains from taking over New York.

After being saved by Scarlet Spider following Arnim Zola's takeover of the S.H.I.E.L.D. Tri-Carrier, Spider-Man must work with him to liberate Manhattan and his fellow students from HYDRA's clutches. Even when they save Agent Venom and Iron Spider, they must also undo transformations of the HYDRA Goblins, avoid Doctor Octopus and Kraven the Hunter, and defeat Arnim Zola.

After Doctor Octopus and Rhino have escaped the Triskelion with Agent Venom in their clutches, Spider-Man and Scarlet Spider track them to the recently-returned HYDRA Island as Doctor Octopus and Arnim Zola work to separate the Venom symbiote from Flash Thompson. In order to get to Flash, Spider-Man and Scarlet Spider have to fight their way past Kraven the Hunter and the HYDRA Agents. Ock Manages to succeed in separating the symbiote from Flash, giving to Kraven, who becomes a lion-like Venom. However, Flash is able to coerce the symbiote back to him as they understand each other. As a result of the re-fusion, Agent Venom gains a new look.

When Doc Ock learns that the key to a device that will retrieve Hydra Island is at Aunt May's house, Spider-Man must fight to protect his family.