Tropical forests are three-dimensional spaces with species and resources heterogeneously distributed. The vertical stratification of tropical forest biotas has been observed for several organisms and regions, but, surprisingly, the vertical structuring of large areas of important tropical forests, such as Brazil's Atlantic Forest, remains poorly studied. Here, we addressed the use of different Atlantic Forest strata by bats, comparing ensemble composition and relative abundance between the understorey and the canopy. A total of 618 bats belonging to 31 species and four families were recorded, including 11 species of frugivores and seven species of gleaning insectivores, the two trophic guilds predominantly represented in our sampling. Fifteen species were captured exclusively in the canopy, and six exclusively in the understorey, and many of those species were represented by a low number of captures (<5). The bat species composition, richness and relative abundance between canopy and understorey strata varied. Chiroderma villosum was exclusively captured in the canopy, Artibeus lituratus was netted predominantly in the canopy and Carollia perspicillata and Desmodus rotundus were mostly captured in the understorey. Although processes such as resource partitioning between species and ecomorphological constraints may explain the differential use of forest strata, this remains little understood because of the scarcity of data for the Atlantic forest canopies.