Bats, along with their ectoparasites, harbor a wide diversity of symbiotic bacterial microorganisms, including potential pathogens. Despite the enormous diversity of bats and associated ectoparasites in Brazil, few studies aimed to investigate the bacterial microbiome of chiropterans and associated flies and mites. This study aimed to characterize the bacterial microbiome of non-hematophagous bats and associated Streblidae flies and Macronyssidae and Spinturnicidae mites in the state of Mato Grosso do Sul, midwestern Brazil.