Understanding effects of flow alteration on stream biota is essential to developing ecologically sustainable water supply strategies. We evaluated effects of altering flows via surface water withdrawals and instream reservoirs on stream fish assemblages, and compared effects with other hypothesized drivers of species richness and assemblage composition. We sampled fishes during three years in 28 streams used for municipal water supply in the Piedmont region of Georgia, U.S.A. Study sites had permitted average withdrawal rates that ranged from < 0.05 to > 13 times the stream’s seven-day, ten-year recurrence low flow (7Q10), and were located directly downstream either from a water supply reservoir or from a withdrawal taken from an unimpounded stream. Ordination analysis of catch data showed a shift in assemblage composition at reservoir sites corresponding to dominance by habitat generalist species. Richness of fluvial specialists averaged about 3 fewer species downstream from reservoirs, and also declined as permitted withdrawal rate increased above about 0.5 to one 7Q10-equivalent of water. Reservoir presence and withdrawal rate, along with drainage area, accounted for 70% of the among-site variance in fluvial specialist richness and were better predictor variables than percent of the catchment in urban land use or average streambed sediment size. Increasing withdrawal rate also increased the odds that a site’s Index of Biotic Integrity score fell below a regulatory threshold indicating biological impairment. Estimates of reservoir and withdrawal effects on stream biota could be used in predictive landscape models to support adaptive water supply planning intended to meet societal needs while conserving biological resources.