Understanding the role of hydrologic variation in structuring aquatic communities is crucial for successful conservation and sustainable management of native freshwater biodiversity. Partitioning beta diversity into the additive components of spatial turnover and nestedness can provide insight into the forces driving variability in fish assemblages across stream flow regimes. We examined stream fish beta diversity across hydrologic and anthropogenic disturbance gradients using long-term (1916–2016) site occurrence records (n = 17,375) encompassing 252 species. We assessed total beta diversity (Sørensen dissimilarity), spatial turnover, and nestedness of fish assemblages in contrasting stream flow regimes across a gradient of decreasing flow stability: groundwater stable (n = 77), groundwater (n = 67), groundwater flashy (n = 175), perennial runoff (n = 141), runoff flashy (n = 255), and intermittent (n = 63) streams. Differences in total beta diversity among the stream flow regimes were driven predominantly (>86 %) by spatial turnover (i.e. species replacement) as opposed to nestedness (i.e. species loss or gain). Total fish beta diversity and spatial turnover were highest in streams with intermediate flow stability (groundwater flashy), while more flow-stable streams (groundwater stable and groundwater) had lower turnover and higher nestedness. Species turnover was also strongly associated with seasonal variation in hydrology across all flow regimes, but these relationships were most evident for assemblages in intermittent streams. Distance-based statistical comparisons showed significant correlations between beta diversity and anthropogenic disturbance variables, including dam density, dam storage volume and water withdrawals in catchments of groundwater stable streams, while hydrologic variables were more strongly correlated with beta diversity in streams with runoff-dominated and flashy flow regimes. The high spatial turnover of species implies that fish conservation actions would benefit from watershed-focused approaches targeting multiple streams with wide spatial distribution, as opposed to simply focusing on preserving sites with the greatest number of species.