In the fourth season, Doctor Octopus allies with Zola and Hydra and forms a new iteration of the Sinister Six consisting of Kraven the Hunter, Ultimate Green Goblin, Rhino, Hydro-Man, and Electro. In response, Spider-Man forms a team consisting of Agent Venom, Iron Spider, Miles Morales, and Scarlet Spider (Ben Reilly). It is later revealed that Ben is a clone of Spider-Man created by Doctor Octopus, but Spider-Man convinces him to reform.
Later, Crossbones and Morbius experiment with Venom, turning New York's citizens into Carnage-like monsters. Carnage possesses Mary Jane Watson, but Spider-Man and Harry Osborn free her. Unbeknownst to Spider-Man, a fragment of the Carnage symbiote remains inside Mary Jane's body.
Madame Web learns that the destruction of the Siege Perilous shattered the barrier between universes. Spider-Man and Miles Morales travel to alternate realities to collect the fragments of the Siege Perilous and battle Wolf Spider, who seeks to reassemble the Siege Perilous to conquer the multiverse. After reassembling the Siege Perilous, Miles moves to the main universe and lets his version of Gwen Stacy replace him.
Spider-Man and Mary Jane later discover a second clone of Spider-Man named Kaine, who tries to feed off them. During this time, Mary Jane learns to control Carnage and becomes Spider-Woman. It is discovered that Doctor Octopus and Arnim Zola created Scarlet Spider as part of Weapon S, a project to create synthezoid Spider-Slayers. Kaine fuses with the synthezoids to become the Ultimate Spider-Slayer, but is defeated by Agent Venom.
In the series finale, the Sinister Six capture Spider-Man and use an antidote to remove his powers. After working with Norman Osborn, Peter regains his powers and defeats the Sinister Six. Spider-Man convinces Doctor Octopus to surrender, after which he and the S.H.I.E.L.D. trainees graduate.
