AbstractSurface water drying challenges the persistence of lotic biological assemblages in non‐perennial streams and rivers. However, the effects of natural hydrological disturbances on an assemblage depend on individual species characteristics. In this study, we investigated the structure and association of fish and macroinvertebrate assemblages during two phases in non‐perennial streams (flowing vs. disconnected pools). We sampled fish and macroinvertebrate assemblages in five non‐perennial stream reaches located in the Caatinga (Brazilian semi‐arid region). Fish species were resistant to hydrological dynamics in the streams. On the other hand, some macroinvertebrate taxa from the flowing phase disappeared, and new taxa colonized the disconnected pools. The absence of lotic insects following flow cessation and the colonization of disconnected pools by lentic taxa facilitated macroinvertebrate assemblages persistence in these dynamic streams. Our study showed that fish and macroinvertebrate assemblages respond differently to the same natural flow cessation, which leads us to predict that flow changes will yield different assemblages, depending on the taxa. Thus, it is crucial to consider multi‐assemblage responses to effectively manage and conserve non‐perennial stream ecosystems in a tropical semi‐arid region.