Scorpion (Jason Spisak) - Scorpion is a criminal that was foiled by Spider-Man when he tried to steal a jewel from a museum. He later took part in Kraven the Hunter's show: "Kraven's Amazing Hunt" to lure Spider-Man and Miles Morales to the site only to be defeated by both Spider-Men.
Battlestar - Lemar Hoskins appears in a non-speaking role, in the season two episode, "Take Two".
Ben Parker (Patton Oswalt) - Uncle Ben appears posthumously in this series. Here, he is depicted as a budding scientist and a clock designer. In a flashback, it is revealed that he reimagined his "With great power comes great responsibility" quote as a scientific formula, which Peter regards as goofy.
Michael Korvac (Wil Wheaton) - This version is a human-shaped alien scientist who was in love with the A.I. Rora until he upgraded himself to android to an A.I. system on his space station ever since Rora was stolen from him by J'son.
Korath the Pursuer (Dave Fennoy) - Korath is a subordinate of Thanos, who was tasked to retrieve the Cosmic Seed and bring it to him.
Thor Odinson - Thor appears in the episode Accidents Will Happen where J'son introduces Peter to the Galactic Council.
Loki Laufeyson
Carnage - The Carnage Symbiote appears in the Guardians of the Galaxy episode "Back in the New York Groove". After Thanos escaped from his underground imprisonment beneath Manhattan, he came across the Carnage Symbiote and bonded to it.