With increased pressure from a growing human population, managers are challenged to understand how novel disturbances (e.g., climate change, increased water withdrawals, urbanization) may affect natural resources. The Sudbury River is a National Wild and Scenic River located in suburban Boston, Massachusetts (Northeastern US) with myriad impairments (e.g., mainstem impoundments, withdrawals, and urbanization) that is under increasing pressure from hydrologic alteration. We sampled fish, mussel, and macroinvertebrate assemblages in the Sudbury River and used species traits to investigate potential effects of past and future flow alteration on biota. Analysis of 33 years of stream gage data indicates continued hydrologic alteration of the Sudbury River, likely related to increased urbanization and water withdrawals over that time. These changes include a roughly 200% increase in rise rates of flows, an approximate 65% decrease in 1-day minimum flows, and a trend towards increasing high flow pulse counts. Biotic sampling in summer of 2014 demonstrated that the Sudbury River is now dominated by generalist species. Of five mussel species sampled, all are generalists in their habitat requirements. Though one mussel species of special concern was sampled, the most abundant species collected were the widespread Eastern elliptio (58%) and Eastern lampmussel (40%). We used the target fish community (TFC) model to assess the degree to which the fish assemblage deviated from that expected for a river with similar zoogeographic and physical features. Overall, the current community has a 22.7% similarity to the TFC. Of the four fluvial specialist species present in the TFC, only fallfish was sampled in our study. While the TFC showed that the historical assemblage was likely dominated by fluvial specialist and fluvial dependent species, the current assemblage is overwhelmingly dominated by macrohabitat generalists (90.6% of fishes sampled). These results are consistent with other studies that show shifts in assemblages from fluvial specialists to habitat generalists with hydrologic alteration. If the current trends continue, it is likely that biotic assemblages will experience increasing pressure from hydrologic alteration. While hydrologic alteration is likely impacting biotic assemblages in the Sudbury River, other factors such as high temperatures, low dissolved oxygen, high nutrients, low availability of high-quality habitat, and poor habitat connectivity may also be negatively impacting biotic assemblages. Comparisons to other rivers and a complete longitudinal habitat survey could help to identify availability of unique habitats and representativeness of this study. While this study suggests impacts of flow on biota, future studies with quantitative, habitat-specific sampling during different flow levels could help to directly identify links between hydrologic alteration and biotic impairment in the Sudbury River.