Although no one on the animation team has directly confirmed it, many viewers interpret Bob as bisexual. As noted in Emma Flint's 2021 Digital Spy article, “Bob's Burgers — Bob Belcher is actually bisexual (and always has been),” people have been examining the character’s sexuality for several years. The most notable instance of Bob talking about his sexuality occured in season 4 episode 5 "Turkey in a Can" in an exchange between Bob and a deli man.

Another family member, Gene, who frequently appears in drag, has also been discussed in terms of representation. In a 2016 Yahoo interview “'Bob's Burger's Creator Loren Bouchard Talks Holidays With the Belcher's, Gene's Feminism, and More Megan Mullaly“ Bouchard addressed Gene's possible gender fluidity in a way that allows for an open interpretation about the character.

There are, however, several canonically queer characters in the film, including Marshmallow, who appears in the end credits dance scene. Originally voiced by David Herman, a cisgender actor, Marshmallow was recast in 2023. Bouchard announced the change in a response on X stating that activist and model Jari Jones would voice the role. Jones officially started portraying the character in the season 14 episode 1 "Fight at the Not Okay Chore-ral."

Another queer character who appears in the film is Little King Trashmouth. His sexuality was first mentioned when Linda talks about him in season 3, episode 17 "Two for Tina." Little King Trashmouth (and sometimes with his husband, Gary) has appeared in the annual Bob’s Burgers Pride merchandise, particularly in the 2021 and 2023 collections.

Brunhilde is arguably one of Bugs Bunny’s most recognizable disguises, appearing on things like postage stamps to a skin option in the online game MultiVersus.