Non-perennial rivers (NPRs) are highly dynamic ecosystems that cease to flow or dry up partially or completely across time and space, representing over half of the global river network. These systems are expanding globally under climate change, yet their biodiversity patterns remain poorly understood in tropical semi-arid regions. We evaluated the effects of seasonal drying on the taxonomic (alpha and beta) and functional diversity of fish and aquatic insects in the Cruxati NPR. Fish alpha-diversity remained stable between the two phases, likely reflecting physiological, trophic, and reproductive adaptations of dominant species. Insects exhibited higher alpha-diversity during the non-flowing phase, driven by lentic-adapted taxa. Fish beta-diversity increased during the non-flowing phase because of environmental heterogeneity across isolated pools and founder effects, whereas insect beta-diversity did not vary, possibly because of the limited spatial scale. Functional diversity remained unchanged between flowing and non-flowing phases in both fish and aquatic insect communities, likely because of historical periods of intense and frequent droughts. Our findings reveal that aquatic communities in semi-arid NPRs exhibit distinct results from those in temperate regions, showing seasonal persistence and highlighting the importance of historical and regional contexts in shaping biodiversity responses to hydrological fluctuations. Given the strong dependence of these communities on remnant isolated pools during the non-flowing phase, proper water resource management is crucial to ensure the ecological resilience of NPRs.