An extensive survey of the ectoparasites infesting bats in Paraguay provides information regarding the taxonomy and host distribution of streblid bat flies at a geographic interface between subtropical and temperate habitats. Five families of bats representing 45 species, including Molossidae (5 genera and 15 species), Natalidae (1 genus and 1 species), Phyllostomidae (11 genera and 15 species), Noctilionidae (1 genus and 2 species), and Vespertilionidae (4 genera and 12 species) were collected from 24 localities across Paraguay and sampled for ectoparasites. In total, 2,467 bat flies were collected, representing 11 genera and 31 nominal species of Streblidae, of which 6 genera and 24 species are new records for Paraguay. No streblids were collected from vespertilionid bats; 23 species infested phyllostomids, 6 species noctilionids, 1 species a natalid, and 1 species molossids. Streblid bat flies were highly specific to certain host groups and individual host species, and their geographic distributions closely followed those of their host bats. Of 31 streblid species surveyed, 27 were monoxenous (i.e., associated with a single host species), and 4 were stenoxenous (i.e., associated with a group of phylogenetically related hosts). The number of streblid species is greatly reduced in the Chaco region west of the Paraguay River, largely because of the lack of phyllostomid host bats.