We compared the assemblages of phyllostomid bats in three Neotropical rainforests with respect to species richness and assemblage structure and suggested a method to validate estimates of species richness for Neotropical bat assemblages based on mist-netting data. The fully inventoried bat assemblage at La Selva Biological Station (LS, 100 m elevation) in Costa Rica was used as a reference site to evaluate seven estimators of species richness. The Jackknife 2 method agreed best with the known bat species richness and thus was used to extrapolate species richness for an Amazonian bat assemblage (Tiputini Biodiversity Station; TBS, 200 m elevation) and an Andean premontane bat assemblage (Podocarpus National Park; BOM, 1000 m elevation) in Ecuador. Our results suggest that more than 100 bat species occur sympatrically at TBS and about 50 bat species coexist at BOM. TBS harbours one of the most species-rich bat assemblages known, including a highly diverse phyllostomid assemblage. Furthermore, we related assemblage structure to large-scale geographical patterns in floral diversity obtained from botanical literature. Assemblage structure of these three phyllostomid assemblages was influenced by differences in floral diversity at the three sites. At the Andean site, where understorey shrubs and epiphytes exhibit the highest diversity, the phyllostomid assemblage is mainly composed of understorey frugivores and nectarivorous species. By contrast, canopy frugivores are most abundant at the Amazonian site, coinciding with the high abundance of canopy fruiting trees. Assemblage patterns of other taxonomic groups also may reflect the geographical distribution patterns of floral elements in the Andean and Amazonian regions. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 617–629.