Abstract Previous research has shown diverse vertical space use by various taxa, highlighting the importance of forest canopy. Yet, we often fail to explore how this three-dimensional space use changes over time. Here we use canopy tower systems in French Guiana to monitor neotropical bat activity above and below the forest canopy throughout nine nights in the wet season. We show that different bats use both canopy and understory space differently, and that this can change throughout the night. We find that bats are overall more active in the canopy, but multiple species/acoustic complexes are more active in the understory. We also find that species that do not seem to prefer understory or canopy, when data are aggregated by night, do show temporally changing preferences in hourly activity. This work highlights the need to consider temporal axes in studies of space use, both throughout daily cycles and across seasons.