Despite a long history of scientific collection of bats, Hispaniola remains the least studied island of the Greater Antilles. Using standardized trapping methods during the wet and dry season at four major caves — Honda de Julián, La Chepa, Los Patos, and Pomier #4, we sampled a total of 1,472 individuals in four families, 11 genera and 12 species (of 18 recorded for the island). We report significantly fewer captures on the second night of sampling. We document seasonal variation in abundance of Macrotus waterhousii, Monophyllus redmani, and Artibeus jamaicensis, that results in 1–4 more species captured in the wet season. Additionally, singleton captures at all caves except for Honda de Julian produced wide confidence intervals in estimates of richness. Finally, we highlight the role of caves as major ecosystems for maintaining Hispaniolan mammal biodiversity. The high diversity recorded at La Chepa, together with possible declines at the historically very diverse Los Patos, highlight the conservation importance of all surveyed caves.