Kruger National Park, South Africa, is home to at least 39 species of Microchiroptera (insectivorous bats). In an effort to gain a better understanding of their population ecology and distribution, we performed an acoustic survey using ultrasonic detection to quantify bat activity and to determine the habitat preferences of each identified species. Throughout 15 microsites, a total of 2398 recorded calls were identified as belonging to nine identified species and two unidentified species. We sampled around two main camps in the park and there was no significant difference found in the number of calls recorded between the Skukuza and Shingwedzi camps. While it is clear that some species frequent some microsites more often than others, a statistical analysis concludes there was no significant different in species-specific microsite preference. Additionally, no significant difference was found in call abundance between disturbed microsites and natural microsites. This work should set the stage for further research on Microchiroptera distributions and habitat preferences in the park. Our findings can be built upon with future research in hopes that, eventually, a complete call library is created and used to monitor and conserve Microchiroptera populations within the park.