- Nadji Jeter - Miles Morales (voice)
- Nadji Jeter - Security Robot (voice)
- Alastair Duncan - Adrian Toomes (voice)
- Alastair Duncan - Thug #3 (voice)
- John DiMaggio - Raymond Warren (voice)
- John DiMaggio - Policeman (voice)
- John DiMaggio - Security Guard/Customer (voice)
- Robbie Daymond - Peter Parker / Spider-Man (voice)
- Robbie Daymond - Bully #1 / Hot Dog Announcer / Ticket Taker (voice)
- Robbie Daymond - Cop #1 (voice)
- Scott Menville - Doc Ock / Otto Octavius (voice)
- Scott Menville - Grady Scraps (voice)
- Scott Menville - The Living Brain / Cop #2 (voice)
- Nancy Linari - Aunt May (voice)
- Nancy Linari - Woman Bystander (voice)
- Nancy Linari - Landlady (voice)
- Nancy Linari - Bomb Voice (voice)
- Max Mittelman - Harry Osborn (voice)
- Max Mittelman - Thug #2 / Little Kid (voice)
- Max Mittelman - Police Officer #1/Osborn Technician #2/Police Officer #2 (voice)
- Max Mittelman - Male Clerk/Guy in Line (voice)
- Laura Bailey - Gwen Stacy (voice)
- Laura Bailey - Tully (voice)
- Laura Bailey - Osborn Recruiter #2 (voice)
- Josh Keaton - Norman Osborn (voice)
- Josh Keaton - Unidentified Voice / Police Officer (voice)
- Josh Keaton - Hologram (voice)
- Josh Keaton - Prison Guard (voice)
- Melanie Minichino - Anya Corazon (voice)
- Melanie Minichino - Chaperone (voice)
- Melanie Minichino - Student #1 (voice)
- Melanie Minichino - Helicopter Pilot (voice)
- Melanie Minichino - Female Clerk (voice)
- Ben Diskin - Flash Thompson
- Ben Diskin - Police Officer #2/Hoodlum #1/Police Officer #4 (voice)
- Ben Diskin - Flash Thompson (voice)
- Ben Diskin - Osborn Recruiter #1 (voice)
- Ben Diskin - Student #2 (voice)
- Fred Tatasciore - Max Modell (voice)
- Fred Tatasciore - Bystander (voice)
- Fred Tatasciore - Thug #1 / Father (voice)
- Fred Tatasciore - Trucker/Mr. Slott (voice)
- Fred Tatasciore - Police Officer #1 (voice)
- Fred Tatasciore - Interviewer (voice)
- Cameron Boyce - Herman Schultz / Shocker (voice)
- Nathaniel J. Potvin - Prowler (voice)
- Richard Doyle - Police Officer #3 (voice)
- Zeno Robinson - Randy Robertson (voice)
- Daniel Hagen - Cop (voice)
- Jason Spisak - Alistair Smythe (voice)
- Jason Spisak - Scorpion / Purse Snatcher / Driver (voice)
- Jason Spisak - Audience Member (voice)
- Jason Spisak - Alistair Smythe / Scorpion (voice)
- Matthew Mercer - Aleksei Sytsevich / Rhino / Reporter (voice)
- Matthew Mercer - Aleksei Sytsevich/Comic Book Guy (voice)
- Grey DeLisle - Black Cat (voice)
- Grey DeLisle - Black Cat/Nurse/Mom (voice)
- Travis Willingham - Sandman / Flint Marko (voice)
- Sofia Carson - Sand Girl (voice)
- Sofia Carson - Keemia (voice)
- Yuri Lowenthal - Nocturnal (voice)
- Yuri Lowenthal - Curt Connors (voice)
- Yuri Lowenthal - Clayton Cole / Clash (voice)
- Jeremy Shada - Ross Caliban (voice)
- Bob Joles - J. Jonah Jameson (voice)
- Aaron Abrams - Tinkerer (voice)
- Aaron Abrams - Tinkerer / Old Man (voice)
- Aaron Abrams - Tinkerer / Police Officer (voice)
- Sumalee Montano - Yuri Watanabe (voice)
- Felicia Day - Mary Jane (voice)
- Alex Désert - Swarm (voice)
- Yvette Nicole Brown - Head Administrator (voice)
- Stan Lee
- Alan Fine
- Joe Quesada
- Dan Buckley
- Jeph Loeb
- Brian Tyler
Robbie Daymond interpreta a Peter Parker / Spider-Man (voice) en la serie.
Nadji Jeter interpreta a Miles Morales (voice) en la serie.
Fred Tatasciore interpreta a Max Modell (voice) en la serie.
Laura Bailey interpreta a Gwen Stacy (voice) en la serie.
Melanie Minichino interpreta a Anya Corazon (voice) en la serie.
Nancy Linari interpreta a Aunt May (voice) en la serie.
Patton Oswalt hace el papel de Uncle Ben (voice).
Scott Menville hace el papel de Doc Ock / Otto Octavius (voice).
Jim Cummings interpreta a Hammerhead (voice) en la serie.
Ben Diskin interpreta a Flash Thompson en la serie.
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