Although water availability is known to affect landscape-scale patterns of wildlife diversity and distribution in arid environments, little is known about the microhabitat characteristics that shape the local-scale distribution of desert bats. We examined the relative importance of pond microhabitat characteristics for the conservation of bats, and hypothesized that in arid environments, patterns of bat diversity and community composition relate to the size of the pond and its hydroperiod (the number of months a pond holds water), a term we use to distinguish between permanent, semi-permanent and temporary ponds. We combined acoustic monitoring with video recording and an experimental approach to study bat activity over natural ponds in the Negev Desert, Israel. We found that both within and between ponds bat species richness and activity significantly increased with pond size. An experimental reduction of pond size led to a significant reduction in bat species richness and activity and affected the bat community composition. In contrast to pond size, pond hydroperiod did not affect bat diversity, as temporary ponds had equivalent levels of bat species richness and activity to permanent ponds. However, hydroperiod did couple with pond size to affect the bat community composition, whereby non-desert bat species that have a higher frequency of drinking were associated with larger and more permanent ponds. Our results highlight the importance of larger temporary ponds (ponds over 15 m in length and 0.5 m in depth) for the conservation of biodiversity in arid environments.