Quantifying assemblage structure across spatio-temporal scales is ecologically important and further aids in the understanding of community organisation processes. Currently, few studies have assessed assemblage structure across generous magnitudes of scale, and influences of processes (biotic and abiotic) responsible for structuring assemblages are still questioned. Using community and hydrologic data collected over a 22-year period from a stretch of river nearing 150 km, we examined spatio-temporal fish assemblage structural patterns in a temperate coastal plain stream. Results indicated that significant changes in assemblage structure across time were influenced by environmental disturbances, including drought and hurricane events. Assemblages were restructured in a punctuated manner directly following these events, and complete recovery of initial assemblage structure did not occur across the study period. Additionally, we found spatial differentiations between upstream and downstream assemblages, which were driven by greater abundances of several species in downstream sites. Our results suggest that assemblage structure is influenced by environmental variation, specifically, extreme disturbance events and spatial habitat heterogeneity.