Human alteration of the planet’s terrestrial landscapes for agriculture, habitation and commerce is reshaping wildlife communities. The threat of land cover change to wildlife is pronounced in regions with rapidly growing human populations. We investigated how species richness and species-specific occurrence of bats changed as a function of land cover and canopy (tree) cover across a rapidly changing region of Florida, USA. Contrary to our predictions, we found negligible effects of agriculture and urban development on the occurrence of all species. In contrast, we found that a remotely sensed metric of canopy cover on a broad scale (25 km2) was a good predictor of the occurrence of eight out of ten species. The occurrence of all smaller bats (vespertilionids) in our study increased with 0–50% increases in canopy cover, while larger bats showed different patterns. Occurrence of Brazilian free-tailed bats (Tadarida brasiliensis) decreased with increasing canopy cover, and Florida bonneted bats (Eumops floridanus) were not influenced by canopy cover. We conclude that remotely sensed measures of canopy cover can provide a more reliable predictor of bat species richness than land-cover types, and efforts to prevent the loss of bat diversity should consider maintaining canopy cover across mosaic landscapes with diverse land-cover types.