A primary focus of ecological research is to characterize the factors responsible for patterns in biodiversity and assemblage structure along gradients of resources and environmental conditions. In addition to biotic and abiotic factors, the spatial relatedness of locations can also influence assemblage patterns and should be accounted for when inferring the influences of environmental factors on assemblage structure. To address this issue, we examined the influences of hydrology, food resources, and a spatial component by comparing assemblage attributes of fishes and macroinvertebrates at 13 sites in the Meramec River watershed, southeastern Missouri, USA. Redundancy analyses and multiple regression techniques were used to predict variation in species composition, richness, and abundance. Results suggest that spatial processes play an important role in structuring fish assemblages, while macroinvertebrates are regulated to a greater extent by local environmental conditions. Additionally, hydrologic variability, alone or in combination with resource abundance and habitat quality, explains considerable amounts of variation in composition, richness, and abundance of fishes and macroinvertebrates. In particular, fish species richness increases with increasing flow variability, potentially because the majority of species are characterized by opportunistic and equilibrium life history strategies, which are favored by hydrologic variability; we also observed negative effects of extreme hydrologic variability on macroinvertebrate taxon richness. Overall, these results demonstrate the importance of natural flow regimes for explaining spatial variation in aquatic assemblages at the within‐watershed scale and point to the strength of a framework integrating hydrologic variability, resource abundance, and spatial relatedness when predicting assemblage patterns in freshwater systems.