Sinister Six is a group of supervillains who serve as the antagonistic faction of Ultimate Spider-Man.
Doctor Octopus' side
- Otto Octavius/Doctor Octopus (William Salyers) - Founder and leader (Deceased, dies of a brain tumor). Evidence: metallic arms.
- Adrian Toomes/Vulture (Bruce Willis) - Second-in-command (Incarcerated). Evidence: Hi-Tech wings.
- Max Dillon/Electro (Phil LaMarr) - Enforcer (Deceased, killed by overloading electricity). He first appears as a former electrician who was working on something but gets electrocuted by it and falls into a water filled with electric eels. When he wakes up in the hospital, he gets scared and leaves the hospital while finding some clothes. While he goes to Times Square, he starts shutting off the power and this gets Spider-Man's attention. Despite not knowing it's Max, he still tries to reason with him but things get worse when police snipers accidentally shoot at Max, causing him to angrily unleash a electrical force. When his screens of are replaced by Spider-Man, he accuses him of being selfish and wanting popularity but Spider-Man unsuccessfully tries to calm him down. Refusing to trust him and feeling set up by him, he fights Spider-Man but gets hit with a fire hydrant and lands into a billboard. However, he takes out two plugs from it and charges up. He then attacks the city with his electricity but is stopped when Spider-Man and some firefighters spray him with water and make him land into a pool. Before he was arrested, he glares at the hero and vows revenge. In prison, he despises the name Max Dillon and renames himself Electro. During a break in, the villains find Electro and break him out. He then joins them and agrees that they have a spider to squish. After Spider-Man recovers from a collapsed warehouse, Electro meets him at the power grids and plans to be like a god to humanity. They then fight but Electro is about to win until Gwen Stacy arrives to help take care of him. Spider-Man fights Electro while Gwen goes to the control room to help stop Electro. After a fight, Peter makes a web field and they shoot electricity at Electro, overloading him. He then turns into a rock statue, breaks apart, and explodes, killing him. Despite them being enemies, Spider-Man and the heroes do a little bit feel sorry for Electro and decide to give him a grave. Evidence room: Battery chip implant.
- Herman Schultz/Shocker (Will Smith) - Enforcer (Incarcerated). Evidence room: One of his shock gauntlets.
- Flint Marko/Sandman (John DiMaggio) - Goon (Missing). Evidence room: Hourglass of sand.
- Alex O'Hirn/Mecha Rhino (Clancy Brown) - Goon (Incarcerated). Evidence room: Hi-Tech Rhino suit.
- Phineas Mason/Tinkerer (Jay Klaitz) - Engineer (Still at large).
- Aaron Davis/Prowler (Phil LaMarr) - Buyer (Defected).
Green Goblin's Sinister Ten
- Norman Osborn/Ultimate Goblin (Norman is played by Ben Mendelsohn and Goblin is voiced by Kevin Michael Richardson) - Founder and leader (Deceased, killed in self-defense by Spider-Man). Evidence room: His helmet (the 2012 Super Shredder helmet).
- Flash Thompson (Matt Lanter)/Anti-Venom (voiced by Matt Lanter) - Symbiote host (Under house arrest). Evidence room: A container of his symbiote.
- Sergei Kravenoff/Kraven the Hunter - Bounty Hunter (Incarcerated). Evidence room: Hunting equipment.
- Dimitri Smerdyakov/Chameleon - Imposter (Incarcerated). Evidence room: Shapeshifting mask.
- Carlton Stand/Electric (voiced by Josh Keaton) - Goon (Unknown). Evidence room: Electronic vest.
- Clifton Shallot/Vulturion (Ben Kingsley) - Burglar (Deceased, killed in crash). Evidence room: Wing suit.
- Macdonald "Mac" Gargan/Scorpion (Michael Pena) - Weapons Buyer (Imprisoned). Evidence room: Scorpion tail.
- Aleksyi Sytsevich/Rhino (voiced by Fred Tatasciore) - Muscle (Imprisoned). Evidence room: Rhino horn.
- Jackson Brice/Montana (Jeff Bennett) - Enforcer (Deceased, accidentally killed by Vulture). Evidence room: Sonic gauntlets.
- Raymond Warren/The Jackal - Scientist (Incarcerated). Evidence room: His experiments.
- Donald Menken/Hobgoblin (voiced by Mark Hamill) - Current crime lord and comic relief (Deceased, killed by Ronin). Evidence room: Glider.