The show definitely does this quite a bit.
- Anya Corazón. There are many, many Spider-People around who are much more well-known and popular than her (though she certainly has her fans), so having her show up is something of a surprise.
- The show features the animated TV series debut of the Jackal, the infamous main antagonist of the infamous Clone Saga. While his civilian identity Miles Warren has appeared in past adaptations, both series ending just before he got to properly ascend to becoming a main threat, this marks the first time his villainous alter-ego has made an appearance.
- To a lesser degree, Max Modell, a supporting character who was created for Dan Slott's run in the comics. Plus, the character is gay, something Disney censors haven't let by until quite recently (though Max's sexuality could just go unacknowledged in the show)note It makes sense when one remembers that his creator is involved in the show.
- Clayton Cole is a recent character from the comics who serves as the minor super villain Clash, so seeing him at all was a bit of a shock.
- After fifteen years since the first Tobey Maguire Spider-Man film, nobody really expected to see the pro-wrestler Bonesaw show up again in one of the Origin Shorts.
- Screwball, a lesser known villain in the comic books, makes an appearance. What's even more surprising is that Liz Allan (a character who is much more well known than she is) is her alter ego, and she went through Adaptational Heroism .
- The Blizzard picked for the series isn't the better-known Donald Gill or Gregor Shapanka, but the third one, Randall Macklin, a One-Shot Character who never even fought Spider-Man in the comics.
- Carolyn Trainer, AKA Lady Octopus, is from a period considered an Audience-Alienating Era in the comics, but appears here.
- Three villains introduced in a Season 2 episode; Hippo, Panda-Mania, and Overdrive, who are complete D-Listers. Hell, Panda-Mania doesn't even have a backstory in the comics and is just a random member of the gang of White Rabbit, who herself is a Joke Character.
- The second Electro, Francine Frye, shows up in the fourth part of the "Bring On The Bad Guys" arc, and she's given a Race Lift to black and roughly Peter's age here .
- Puma, a Spider-Man supporting character who was more prominent in the 1980's and 90's, makes his television debut in the episode "Take Two."
- The series implements The Living Brain, a mostly forgotten member of Spider-Man's Rogues Gallery, albeit as another identity for Doctor Octopus.
- "Road to Goblin War" features the TV debut of D-List villain Slyde, who's only appeared sporadically since his introduction in 1986.
- Aside from the 2018 Venom movie where Riot made an appearance, the most prominent symbiotes who appear in Spider-Man and Venom-related TV and film media tend to be either Venom or Carnage, and in more recent times, Agent Venom or Anti-Venom. Beyond that, more minor symbiote characters haven't made many appearances in Spider-Man cartoons and with that in mind, it can come as not-an-unwelcome surprise to see Scream, Scorn, and Mania finally appear... particularly Scream since she's been around since the early 90s for almost as long as Carnage but hasn't really caught on in most Spider-Man media outside of some video games and The Amazing Adventures of Spider-Man.
