Trick or Treat Scooby-Doo! does not include any identifiable transsexual characters or themes. The narrative focuses on a typical Scooby-Doo mystery involving ghosts and villains, without engaging with transgender identity or related topics.

The film primarily features mystery-solving, chases, and the unmasking of villains. No female characters are depicted engaging in or winning direct physical combat against one or more male opponents through skill, strength, or martial arts.

The film features the established Scooby-Doo characters—Fred, Daphne, Velma, Shaggy, and Scooby—all retaining their historically canonical genders. No legacy characters from previous installments or source material have their gender altered.

The film features the established Scooby-Doo characters, all of whom retain their long-standing canonical racial portrayals. No character previously established as one race is depicted as a different race in this installment.