A total of 2075 captures of 11 taxa of bats was recorded during an extensive survey of the vertebrate fauna of the semi-arid Mallee region of Victoria. A further two species, Pteropus scapulatus and Saccolaimus flaviventris, are known from previous records, thus bringing the total to 13 taxa known from the region. There was a marked seasonality in activity patterns and in reproduction. Activity, as revealed by trap captures, peaked over the spring to early autumn months when daytime temperatures are high and nights are mild. Births occurred from October to December, lactating females were recorded from November to February, and juveniles were trapped between December and late February, with minor variation in timing between species. Morphometric measurements revealed that females were generally larger and heavier than males. There was a high level of overlap of species between broad vegetation types. Woodland habitats, especially Riverine Woodland, tended to have a higher frequency of capture and a greater species richness of bats per trapping event than did Mallee Shrubland. The assemblage of bats in the Mallee region, Victoria, like those in other semi-arid regions of southern Australia, includes species that are widespread in Australia (e.g. Chalinolobus gouldii and Nyctophilus geoffroyi), together with species that primarily occur in semi-arid and arid environments (e.g. N. timoriensis, Scotorepens balstoni and Vespadelus baverstocki). This region, which includes mesic riverine habitats, also supports a group of species that are characteristic of temperate south-eastern Australia (e.g. C. morio, V. regulus and V. vulturnus). In comparison with assemblages from temperate and tropical environmental regions, those from the semi-arid region tend to have a lower species richness with fewer families represented, a higher level of insectivory, and a smaller modal body size. The conservation status of bats from the Mallee region, Victoria, is believed to be secure, although the status of N. timoriensis warrants further attention.