Mystique - Mystique's name can be seen on one of the news tickets, noted being questioned in a string of bank robberies. Spider-Man imagines the musical "Mutant and the Beast" starring Mystique and Beast.
Beast - In Ultimate Spider-Man, Spider-Man imagines the musical "Mutant and the Beast" starring Mystique and Beast.
Juggernaut
Liz Allan - Despite not appearing in the TV series, Liz appears in the series's tie-in comic book series. She appears in a small arc watching a football match alongside Mary Jane Watson, and becomes tearfully devastated when the mascot of Midtown High's football team goes missing.
Blade (Terry Crews)
H.E.R.B.I.E.
Batroc (Rob Paulsen) - One of Batroc's robberies is thwarted by someone who looks like Spider-Man. Batroc accepts his defeat and surrenders to go to jail, but "Spider-Man" viciously beats him and Batroc must be hospitalized. Sometime later, when Electro caused a general shutdown, Batroc tried to take advantage of it to steal, but he was stopped by Spider-Man and his team.
Michael Korvac (James Marsters) - Korvac was an intergalactic warlord who led the Chitauri in their conquest. Taking notice of the powerful figures on the planet Earth, including the Avengers, Fantastic Four, and Doctor Strange, Korvac set out to destroy the planet before those heroes had a chance to stop him. The Guardians of the Galaxy, with the help of Nova and Spider-Man, were able to stop his diabolical plan, destroy his flagship, and kill Korvac in the process, putting an end to his reign of terror.
Phil Coulson (Clark Gregg) - Coulson is a member of S.H.I.E.L.D., and working undercover as the principal of Peter Parker's high school. He is also shown wearing tidey whities in the series.
Scarlet Witch - The Super Hero Squad Show version of Scarlet Witch makes a cameo appearance in the episode "Flight of the Iron Spider".
Ben Parker (Greg Grunberg) - Spider-Man has referenced Uncle Ben's death a couple of times when talking about his morals. Ben appeared in the episode "Strange Days" where Nightmare tries to use the failure of Peter being unable to save his uncle's life against him, but it doesn't work. Peter's even grateful that he gets to see Ben again as his uncle encourages him to take the villain down. He was voiced by Greg Grunberg.
Howard Stark (Stephen Collins; older, Charlie Schlatter; young) - Howard Stark appeared in the Avengers Assemble episode "New Year's Resolution", where he and Peggy Carter travelled to the present time and met Tony.
Heimdall (James C. Mathis III AA, Chris Bosh H&TAOS)
Grim Reaper - Grim Reaper fought alongside Crossbones against Cap and Iron Man and then appeared to take Iron Man prisoner. They were then followed by Captain America, who then took out the two villains with Iron Man and took their costumes to go undercover with M.O.D.O.K., Red Skull, and their cabal. Reaper and Crossbones were then apparently beaten by Thor Odinson and Hulk for answers.
Kate Bishop (Ashly Burch) - Kate Bishop makes a cameo in the thirteenth episode of the third season "Into the Future". She resides in a dystopian world in the 30th century and is part of a rebellion to take down Kang.
Justin Hammer (Jason Spisak) - He appears as a wannabe supervillain. He created the robot Adaptoid Android to take on the Avengers. He also mined Vibranium in the Savage Lands and acquired an army of Raptors to fight the Avengers.
Surtur (Rick D. Wasserman) - Thor was sent to Muspelheim when Nighthawk activated the contingency plan against Thor, during which a Norn Stone was attached to the God of Thunder's hammer, causing him to teleport through different realms, one of them being Muspelheim, where he saw Surtur. After being freed from the contingency plan by the Hulk, Thor mentioned that he flew through Muspelheim a total of 81 times. When the Bifrost was affected by the use of the Space Stone, Thor, Hulk, and Hawkeye were accidentally transported to Surtur's realm. They were extracted out of Muspelheim by Heimdall's portal shortly before falling on Surtur's grasp.