| Shameik Moore | Miles Morales / Spider-Man (voice) |
| Jake Johnson | Peter B. Parker / Spider-Man (voice) |
| Hailee Steinfeld | Gwen Stacy / Spider-Woman (voice) |
| Mahershala Ali | Uncle Aaron / Prowler (voice) |
| Brian Tyree Henry | Jefferson Davis (voice) |
| Lily Tomlin | Aunt May (voice) |
| Lauren Vélez | Rio Morales (voice) |
| Zoë Kravitz | Mary Jane (voice) |
| John Mulaney | Peter Porker / Spider-Ham (voice) |
| Kimiko Glenn | Peni Parker (voice) |
| Nicolas Cage | Peter Parker / Spider-Man Noir (voice) |
| Kathryn Hahn | Olivia 'Liv' Octavius / Doc Ock (voice) |
| Liev Schreiber | Wilson Fisk / Kingpin (voice) |
| Chris Pine | Peter Parker / Spider-Man (voice) |
| Natalie Morales | Miss Calleros (voice) |
| Edwin H. Bravo | Brooklyn Visions Security Guard (voice) |
| Oscar Isaac | Interesting Person #1 (voice) |
| Greta Lee | Interesting Person #2 (voice) |
| Stan Lee | Stan (voice) |
| Jorma Taccone | Green Goblin / Last Dude (voice) |
| Joaquín Cosío | Scorpion (voice) |
| Marvin 'Krondon' Jones III | Tombstone (voice) |
| Kim Yarbrough | Scientist In Cafeteria (voice) |
| Lake Bell | Vanessa Fisk (voice) |
| Jessica Mikayla Adams | Brooklyn Friend (voice) |
| Gredel Berrios Calladine | Brooklyn Friend (voice) |
| Sarah D. Cole | Brooklyn Friend (voice) |
| Kelby Joseph | Brooklyn Friend (voice) |
| Mimi Davila | Brooklyn Visions Student (voice) |
| Claudia Choi | Brooklyn Visions Teacher (voice) |
| Melanie Haynes | Brooklyn Visions Teacher (voice) |
| Joseph Izzo | Brooklyn Visions Teacher (voice) |
| Nick Jaine | Brooklyn Visions Teacher (voice) |
| Muneeb Rehman | Brooklyn Visions Teacher (voice) |
| Carlos Zaragoza | Brooklyn Visions Teacher (voice) |
| Post Malone | Brooklyn Bystander (voice) |
| David Applebee | Additional Voices (voice) |
| Juan Carlos Arvelo | Additional Voices (voice) |
| Adam Brown | Additional Voices (voice) |
| Jon Bruno | Additional Voices (voice) |
| Darcy Rose Byrnes | Additional Voices (voice) |
| Oscar Camacho | Additional Voices (voice) |
| June Christopher | Additional Voices (voice) |
| Alycia Cooper | Additional Voices (voice) |
| Michelle Jubilee Gonzalez | Additional Voices (voice) |
| Terrence Hardy Jr. | Additional Voices (voice) |
| Bridget Hoffman | Additional Voices (voice) |
| Rif Hutton | Additional Voices (voice) |
| Miguel Jiron | Additional Voices (voice) |
| Harrison Knight | Additional Voices (voice) |
| Lex Lang | Additional Voices (voice) |
| Donna Lynn Leavy | Additional Voices (voice) |
| Andrew Leviton | Additional Voices (voice) |
| Caitlin McKenna-Wilkinson | Additional Voices (voice) |
| Scott Menville | Additional Voices (voice) |
| Christopher Miller | Additional Voices (voice) |
| Arthur Ortiz | Additional Voices (voice) |
| Juan Pacheco | Additional Voices (voice) |
| Devika Parikh | Additional Voices (voice) |
| Shakira Ja'nai Paye | Additional Voices (voice) |
| Courtney Peldon | Additional Voices (voice) |
| Chrystee Pharris | Additional Voices (voice) |
| Jacqueline Pinol | Additional Voices (voice) |
| Juan Pope | Additional Voices (voice) |
| Al Rodrigo | Additional Voices (voice) |
| Joseph Sanfelippo | Additional Voices (voice) |
| Justin Shenkarow | Additional Voices (voice) |
| Dennis Singletary | Additional Voices (voice) |
| Warren Sroka | Additional Voices (voice) |
| Melissa Sturm | Additional Voices (voice) |
| Holly Walker | Additional Voices (voice) |
| Jason Linere-White | Additional Voices (voice) |
| Ruby Zalduondo | Additional Voices (voice) |
| Ruth Zalduondo | Additional Voices (voice) |