Bioacoustics can be a non-invasive, cost-effective way of studying echolocating bats, and is especially useful for detecting and identifying rare or cryptic species. The insectivorous bats of Madagascar are understudied in comparison to the rest of the island's fauna, and very little is known about their habitat use. Here, we used a remote bioacoustic surveying technique in a bat survey of the Sahamalaza-Îles Radama National Park, northwest Madagascar, to study constant frequency echolocating bats (Hipposideridae and Rhinonycteridae). We used two passive acoustic monitoring units to automatically record bat activity from dusk until dawn in a range of habitat types that are characteristic of the region. Analysis of call acoustic parameters revealed three distinct constant-frequency phonic types within the national park, which we identified as Commerson's leaf-nosed bat (Macronycteris commersoni), the red trident bat (Triaenops menamena) and an unknown phonic type of ca. 78 kHz. We found significant differences in the habitat usage of these three species, suggesting species-specific habitat preferences among Madagascar's bats. Our statistical analyses revealed significant differences between the acoustic echolocation calls of these three phonic types. The 78 kHz calls do not match any of the other constant frequency-calling bat species currently described for Madagascar, indicating either acoustic divergence among bat sub-populations or the possible existence of a new undescribed species. These results highlight the need for increased survey efforts to gain an understanding of species-specific geographic distributions and habitat usage among Malagasy bats and to disentangle their cryptic species complexes.