The most personal enemy for Miles, and arguably most fearsome, Prowler is introduced as a deadly threat who often lurks in the shadows, and sports a set of sharp metal claws that can extend into lasers. Over the course of the film, Miles discovers the villain is actually his uncle Aaron Davis, who became estranged from his brother Jeff after turning to a life of crime.
This incarnation is the film's most direct adaptation of a villain, drawing inspiration from the Ultimate version of the character introduced in 2011 in Ultimate Comics: Spider-Man #1 by Brian Michael Bendis and Sara Pichelli. Voiced by Mahershala Ali, this version is slightly more noble than his comic book counterpart, refusing to kill Miles after discovering his nephew is the new Spider-Man, only to be murdered himself by Kingpin. His death casting a heavy shadow over Miles and his father for the remainder of the film.
While not a villain, per se, Alchemax appears prominently as a nefarious company run by Wilson Fisk to develop the particle accelerator. Much of the action takes place in company's testing facility in Brooklyn, but Miles and Peter also break into a compound in the Hudson Valley to obtain more data about the accelerator, which leads to a spirited chase by trigger-happy scientists after they catch Peter stealing a bagel from the cafeteria.
The organization was introduced by Peter David and Rick Leonardi in Spider-Man 2099 #1 as a mega corporation controlling virtually every aspect of consumer life in the United States. The company was later brought into the Marvel Universe's relative present day by Dan Slott and Ryan Stegman in Superior Spider-Man #19 after a merger by Oscorp and Horizon Labs. Given one character's appearance in the film, Into the Spider-Verse will likely not be the last audiences see of the company.
The most original reimagining of any character is the film's version of longtime Spider-Man foe Doctor Octopus. Introduced as Olivia Octavius, the villain is the lead scientist on the particle accelerator project for Alchemax due to her advanced theories on the existence of the multiverse. After catching Peter Parker in her office attempting to steal data on the project, she reveals herself as Miles' universe incarnation of the classic villain, with her own set of robotic arms.
While the original Doctor Octopus was introduced by Stan Lee and Steve Ditko in The Amazing Spider-Man #3, a female version known as Lady Octopus, or Doctor Octopus II, debuted in The Amazing Spider-Man #406, by J.M. DeMatteis and Angel Medina. Voice by Kathryn Hahn, Into the Spider-Verse's Doc Ock is ultimately taken down by a runaway bus in the extra-dimensional vortex unleashed by her own accelerator.
The last villain to receive a full introduction in the film, Scorpion joins the fight after tracking Miles to Aunt May's home in Queens. More cybernetically enhanced than traditional depictions, this version features a powerful claw arm and piercing tail, and can swap out his legs from a full robotic array befitting his arachnid namesake. Voiced by Joaquin Cosio, this version of the character also appears to be a native Spanish speaker, heard conversing in the language, with translations provided by handy panel on screen.
Introduced by Stan Lee and Steve Ditko in The Amazing Spider-Man #19, this incarnation of the villain puts up a strong fight in the finale, using his tail to disable Peni Parker's trusty SP//dr mech before being dispatched by Spider-Man Noir and Spider-Ham.
Directed by Bob Persichetti, Peter Ramsey and Rodney Rothman, Spider-Man: Into the Spider-Verse stars Shameik Moore, Brian Tyree Henry, Jake Johnson, Mahershala Ali, Hailee Steinfeld, Liev Schreiber, Luna Lauren Velez, Lily Tomlin, Nicolas Cage, John Mulaney and Kimiko Glenn. The film is in theaters now.