D.E.B.S. centers on a romantic relationship between two women, Amy and Lucy Diamond. The narrative portrays their love with humor and genuine affection, as Amy grapples with her feelings for the supervillain Lucy. The film culminates in the affirmation of their relationship, presenting a positive and validating depiction of queer identity and love within a comedic action framework.

The film features an elite team of female secret agents who frequently engage in close-quarters combat. These agents utilize martial arts and physical prowess to defeat multiple male opponents in various action sequences throughout the narrative.

The character Dominique, originally portrayed as white in the source short film, is depicted as East Asian in the feature film. Additionally, the character Janet, who was Black in the short film, is portrayed by a white actress in the feature film.

The film's central narrative celebrates a same-sex romantic relationship, directly endorsing alternative partnership structures over traditional heterosexual marriage. It also features strong female characters operating independently, challenging conventional gender roles within a family context.