Inventories of cave species and in-depth understanding of cave ecosystems are essential for informing conservation approaches for the unique and vulnerable cave fauna. Gcwihaba cave is the largest cave in Botswana but its ecology is poorly understood. This study set out to provide the first quantitative survey of the cave’s terrestrial macroinvertebrates. Macroinvertebrates were collected from sample sites at 10 m intervals into the cave from the cave entrance. At each site, macroinvertebrates on the cave floor were collected by quadrat sampling while macroinvertebrate from cave walls were collected by visual opportunistic searches. Moisture content, pH and electrical conductivity of the cave floor substrate were measured at each site to examine the influence of the floor properties on the distribution of macroinvertebrates on the cave floor. Twelve species in 10 families and 8 orders of terrestrial macroinvertebrates were collected. The occurrence of taxa varied across the sites, with most taxa occurring in the light and twilight sectors of the cave (within 30 m), whereas the dark sector (beyond 30 m) was dominated by cave cockroaches (Gyna sp.). The abundance of the cave cockroaches, darkling beetles (Tenebrionidae, Tenebrio sp.) and cave wasps (Sphecidae) positively correlated with floor substrate of high moisture content and high electrical conductivity, which became increasingly common with distance into the cave. The abundance of other taxa from the cave floor positively correlated with a floor substrate of high pH and low moisture, which was common near the cave entrance.