Water resource managers are challenged to balance growing water demand with protecting aquatic ecosystems and biodiversity. Management decisions can benefit from improved understanding of water withdrawal impacts on hydrologic regimes and ecological assemblages. This study used Ecological Limit Functions for fish groups within the Tennessee and Cumberland river basins to predict species richness responses under simulated constant-rate (CR) and percent-of-flow (POF) withdrawals and for different minimum flow level protections. Streamflow characteristics (SFC) and richness were generally less sensitive to POF withdrawals than CR withdrawals among sites, fish groups, and ecoregions. Species richness generally declined with increasing withdrawals, but responses were variable depending on site-specific departures of SFCs from reference conditions, drainage area, fish group, ecoregion, and minimum flow level. Under POF withdrawals, 10% and 20% daily flow reductions often resulted in loss of <1 species and/or ≤5% richness among fish groups. Median ecological withdrawal thresholds ranged from 3.5-31% for POF withdrawals and from 0.01-0.92 m3/s for CR withdrawals across fish groups and ecoregions. Application of minimum flow level cutoffs often resulted in damping effects on SFC and richness responses, indicating that protection of low streamflows may mitigate hydrologic alteration and fish species richness loss related to water withdrawals. Site-specific and regionally summarized responses of flow regimes and fish assemblages under alternative withdrawal strategies in this study may be useful in informing water management decisions regarding streamflow allocation and maintaining ecological flows.