Bats are a highly diverse order with substantial economic and ecological value. Similarly, forests in South Africa form a valuable biome supporting unique biotic diversity, yet forest bat communities have not previously been surveyed. We sampled 17 forests, of seven forest types and three forest groups, in the Eastern Cape and southern KwaZulu-Natal provinces, located within the Maputaland-Pondoland-Albany global biodiversity hotspot. We utilized capture and acoustic survey methods to compile the first bat species inventory for these forests. Species identification was performed with a combined approach of morphology, echolocation, and DNA barcodes. With this we contributed novel DNA barcodes to the Barcode of Life Data System. A total of 25 species was recorded, with range extensions southward into the Eastern Cape for six species, indicating the region to be more diverse than previously thought. Updated modelled distribution maps for these species are presented. We compiled the first reference call library of hand released bats for South African forest habitats, and the southeast region of the country. We compared species richness, diversity, and dissimilarity of the forest types and found that Scarp forests host the highest species richness and diversity. Patterns of species assemblage similarities between forest groups are attributed to forest biogeographical history and historical dispersal routes of forest fauna. A comprehensive survey, such as this, may assist in the compilation and implementation of forest conservation management plans and future monitoring programs.