Bats play crucial ecological and economic roles. However, this group of mammals is largely threatened due to anthropogenic activities inside or around their caves. In the present study, we investigate the effects of cave microclimate and physiological parameters on bat roost preference in Fairy Cave Nature Reserve (Fairy Cave NR). The microclimate and physiological parameters including temperature (°C), relative humidity (RH), light intensity (lux), air flow, passage dimension (m), roost height (m) and distance of roost from nearest entrance (m) were measured. Results showed that Emballonura monticola, Hipposideros diadema, Hipposideros larvatus and Penthetor lucasi roost in Fairy Cave NR. These bats can be distinguished by their colony size, roost posture, spatial arrangement and position of their roost. Penthetor lucasi makes up the largest colony in the bright zone, whereas E. monticola has the smallest colony, occupying the twilight zone throughout the cave. Members from the family Hipposideridae roost in the dark portion of the cave with zero light intensity. Emballonura monticola utilizes the hottest roost in the cave compared to the other microbats, whereas H. diadema inhabits the coolest roost. Physiological parameters such as light intensity, passage dimension, and distance from nearest entrance are significant parameters in roosting preferences. These parameters should be monitored to ensure the sustainability of not only the aforementioned species, but also other species that roost in nearby caves of Bau limestone (Wind Cave Nature Reserve: with 13 recorded species).