As part of the world biodiversity hotspot 'Meso-America', the country of Guatemala hosts a minimum of 100 bat species. However, the taxonomy and the systematic of Neotropical bats have recently undergone many changes and the knowledge about species diversity and distribution remains patchy. We report here the results of a bat survey conducted in November 2010 inside the biological station 'Las Guacamayas' ('Laguna del Tigre' National Park'). We used three survey methods: roost search, mist net capture as well as ultrasound recording. On the basis of recent taxonomic knowledge, we surveyed a minimum of 25 bat species belonging to seven different families, including two new families for the National Park (Natalidae and Vespertilionidae). We have identified several systematic and taxonomic changes: Carollia brevicauda for C. sowelli, Pteronotus parnellii for P. mesoamericanus, Rhogeesaa tumida for R. bickhami / menchuae, Sturnira lilium for S. parvidens and Uroderma bilobatum for U. convexum. Mist net captures and ultrasound recordings resulted in the identification of several new species. By combining the two inventories of the National Park and taking into account the taxonomic changes, we obtained a maximum of 31 species identified so far. Our survey improves the knowledge on bat diversity and distribution in Guatemala, with implications for their conservation.