We describe levels of parasitism of ectoparasitic bat flies (Hippoboscoidea: Streblidae) on male and female bats from an extensive Neotropical survey. The collection resulted from coordinated vertebrate-parasite surveys undertaken by the Smithsonian Venezuelan Project (SVP) from 1965–1968, which sexed 24 978 bats of 130 species. Streblid parasites were recovered from 6935 individuals of 87 bat species, but only 47 species were captured frequently enough (≥ 20 infested individuals) to permit reliable estimates of streblid parasitism on males and females. Well-sampled species included 39 phyllostomids, four mormoopids, two noctilionids, one natalid and one molossid. Prevalence of streblid parasitism (proportion of individuals infested) of male and female bats was generally not significantly different, and averaged 0.34 across infested species. In species-level analyses assessed against captures, significant sex differences in infestation levels were noted in six species; all had mean prevalence below 0.5 and females were parasitized disproportionately in each. Sex differences in total numbers of flies were noted in 21 species, and in 16 of these, females carried disproportionately heavy loads. Sex differences were also found for eight species of bat in the number of fly species infesting an individual; seven of eight showed heavier female parasitism. In analyses weighted by infestation levels, sex differences in total number of flies were found in only 12 species, with seven showing excessive parasitism of females, and no species showed sex differences in the number of fly species infesting them. These significant biases were not associated with sexual size dimorphism among the bat species. Generally higher levels of parasitism among female bats accords with theory, given their generally higher survivorship and enhanced probabilities of lateral and vertical transmission of host-specific parasites, but contrasts with patterns shown by many other parasitic arthropods. Future analyses should target social groupings of bats, not passively sampled foragers, to better address the mechanisms responsible for this pattern.