Globally, insectivorous bats are vital for maintaining ecosystem function. Sixty-five of the 69 documented South African bat species are insectivorous and are integral to most, if not all, southern African ecosystems. Grasslands cover ca. 29% of South Africa's land area, yet the associated bat biodiversity is understudied. Telperion Nature Reserve (TNR) is a grassland-dominated reserve straddling the border between the Mpumalanga and Gauteng provinces of South Africa, and the grassland and savanna biomes. Using data collected by physical capture and acoustic sampling, an insectivorous bat species inventory for TNR was compiled and a regional echolocation call library was developed. Sixteen sites on the reserve were acoustically sampled for two consecutive nights seasonally (128 sampling nights). Physical capture devices including mist nets, harp traps, and hand nets were used to capture bats. This combination of techniques resulted in the expansion of the insectivorous bat species list for TNR from 13 to 22 species (representing seven families). One hundred and sixty-seven bats from 11 formally recognised species were physically captured. Passive acoustic sampling using full spectrum ultrasonic detectors facilitated the identification of 21 species. Species accumulation curves from acoustic data reached saturation after ca. 40 detector nights. Release recordings from captured individuals were used for the development of the regional call library. This study provides a valuable resource for future bat studies in the grassland biome of South Africa. Furthermore, it expands on previous findings of geographic- and intersex variation in echolocation call parameters of South African rhinolophoid and vespertilionid species, respectively. This study demonstrates and quantifies the minimum effort required to evaluate the bat assemblage composition in southern African grasslands upon which to base regional call libraries. Additionally, it highlights the importance of treating sexes separately when conducting acoustic analysis of bat vocalisations.