Among mammals, bats harbour the greatest taxonomic diversity of ectoparasitic arthropods. This is mainly due to their high mobility, wide distribution range and gregarious social behaviour. In Chile, 17 species of bats have been reported; however, their ectoparasitic arthropofauna has been little studied. There are currently 12 taxa recorded, belonging to two classes and associated with only four species of bats. This study aimed to investigate the diversity of parasitic flies associated with bats in three ecoregions Atacama Desert, Chilean Matorral and Valdivian temperate forest. During 2021, using mist nets, bats were captured in Anzota caves (Atacama Desert ecoregion), Huelquén and Alto Jahuel (Chilean Matorral ecoregion), Cherquenco and San Patricio (Valdivian temperate forest). Each bat was carefully checked during a 15-min interval for the collection of ectoparasites. The arthropods were deposited in vials with 96% ethanol. A total of 26 bats corresponding to three species (Vespertilionidae Histiotus magellanicus Philippi, 1866, Myotis arescens (Osgood, 1943); Phyllostomidae Desmodus rotundus (Geoffroy, 1810)) were captured from which a total of 142 ectoparasitic arthropods were collected. Bat flies were separated/identified under a stereomicroscope. Additionally, from the fieldwork, we report the presence of other ectoparasites associated with Chilean bats. In our study, we report new host-parasite associations between Trichobius parasiticus Gervais, 1844 (Diptera Streblidae) on D. rotundus, and Basilia silvae (Brèthes, 1913) (Diptera Nycteribiidae) in M. arescens in Chile. Our study extended the latitudinal range of distribution for B. silvae to Araucanía region, and we report for first time T. parasiticus in the country. Additionally, partial sequences of the cytochrome c oxidase I gene were obtained from these specimens. Although there is slight morphological variation in the specimens of T. parasiticus, phylogenetic analyses suggest that they correspond to the same species. The sequences generated for B. silvae represent the first for the species. Authors recommend the use of an integrative approach in the identification of ectoparasites in poorly studied ecoregions and hosts. The integration of different markers is necessary to determine more precisely the phylogenetic relationships between South American populations and species of the genera Basilia and Trichobius.