Few studies have focused the vertical structure of bat assemblages, and how it influences community composition. The goal of this study was to analyze the vertical structure of an assemblage of bats in a forest fragment in southern Brazil. Bats were sampled using mist-nets placed at three heights (understory, below-canopy, and canopy). Forest strata were compared with respect to their species richness and diversity. The latter was estimated using the Shannon-Wiener index (H'), and the statistical significance of differences among strata was assessed using t tests. We used an index of Constancy (C) to determine the frequency of a given species in each vegetation stratum, such that a species was considered as "frequent" (C > 50), "less frequent" (25 < C < 50) and "occasional" (C < 25). We captured 485 bats belonging to two families and 24 species. In the understory layer, we captured 173 individuals in 13 species, which resulted in a diversity index of H' = 1.981. In the under-canopy, 153 individuals were caught in 18 species and the resulting diversity index was H' = 2.509. Finally, in the canopy, 159 bats were caught, in 22 species, with the resulting diversity index of H' = 2.442. In the understory and in the canopy, only one species Artibeus lituratus (Olfers, 1818) was classified as "frequent." Four species A. lituratus, Sturnira lilium (É. Geoffroy, 1810), Anoura geoffroyi Gray, 1838, and Eptesicus diminutus Osgood, 1915 were classified as "less frequent" in the under-canopy stratum. All other species recorded in each stratum were classified as "occasional." The studied bat assemblage showed vertical stratification, with the higher strata harboring increased diversity. Our study shows how important it is to sample the upper levels of a forest fragment to obtain a more representative understanding of the use of space by a bat assemblage.