Abstract Balancing aquatic conservation and water supply is becoming a major global issue for urban landscapes. The purpose of this study was to evaluate the ecological effects of stream-flow alterations via water withdrawals and impoundments on fish assemblage structure. Electric fishing data were collected at 33 wadeable streams in Connecticut, located in the Southern New England region of USA. Fish sampling was conducted directly downstream of water withdrawals for municipal and agricultural water supply, and study sites differed in potential water withdrawal rates and the presence of impoundments. Regression analysis showed that water withdrawal rate was more important than other natural and anthropogenic factors (e.g. landcover and stream size) in explaining several fish assemblage metrics. Stream sites with high withdrawal rates were generally characterised by lower proportions of fluvial dependent fishes (fish which need flowing water to complete a portion of their life history) and benthic invertivores (fish which feed on bottom-dwelling stream insects in riffle habitat), and had a greater percent composition of macrohabitat generalists, particularly members of the family Centrarchidae. Some assemblage metrics responded linearly with increasing magnitude of water withdrawals, but others were non-linear. Results are consistent with ecological theory that alteration of the natural flow regime will impact stream biota.