Abstract In the Yucatan, cenotes or water sinkholes formed by the dissolution of limestone and surrounded with a characteristic dense layer of heterogeneous vegetation, are the main sources of water for plant and animal assemblages. We investigated their importance to bats by comparing the assemblage structure and diversity between tropical forest and pastureland, with and without cenotes. We set ground level mist nets, sub-canopy nets and harp traps for 96 nights and caught 2819 bats of 26 species and six families. Forest had a greater bat species diversity and abundance than pastureland, as well as rare and threatened species. In forest, sites with and without cenotes had a similar bat assemblage structure and diversity, whereas in pastureland there was a greater species diversity and abundance and number of rare and threatened species at cenotes than sites without cenotes. Chrotopterus auritus and Micronycteris schmidtorum, both threatened in Mexico, and Eptesicus furinalis, were exclusively captured at cenotes. We suggest that it may be the vegetation around cenotes rather than the water bodies per se that attracts many bat species in pastureland. Cenotes are threatened by nitrate contamination from untreated domestic and animal waste and fertilizers and the removal of vegetation to allow livestock access to water. In Yucatan’s agricultural landscapes, protection of cenotes and their characteristic vegetation is key not only for bat conservation but also because they represent potential seed sources for the regeneration of the lowland forest, in which bats play key roles as pollinators and seed dispersers.