Bats represent a key group in tropical forest dynamics, given their participation in ecological interactions that lead the regulation of these forests. They are also sensitive to the heterogeneous vertical gradient in the forest, called stratification. In this study we evaluated the influence of two different forest strata on species composition and bat guild structure. The samplings were carried out over eight nights in a forest fragment located in the southwest of the Amazon; we used mist nets installed in the understory and sub-canopy. A total of 197 captures were distributed in 19 genera and 25 species; they were all representatives of the family Phyllostomidae. In the sub-canopy, 54 individuals and 15 species were captured, with four exclusive species. In the understory, 143 individuals of 21 species were recorded, of which 10 were exclusive of this stratum. The sub-canopy presented a diversity index greater than the understory, with differences between species composition of the two assemblies, due to the presence or absence of some species. We also found a variation in the presence of frugivorous, insectivorous and omnivorous species, which is the result of differences in the foraging methods of these species and also of the habitat preference. Differences were verified in the assemblies studied, demonstrating the effects of vertical stratification on the bats in the studied fragment. Studies that consider more than one vertical stratum in tropical forests are more representative than sampling with only understory mist nets, given the capture of exclusive species.