Biological communities in freshwater streams are often impaired by multiple stressors (e.g., flow or water quality) originating from anthropogenic activities such as urbanization, agriculture, or energy extraction. Restoration efforts in the Chesapeake Bay watershed, USA seek to improve biological conditions in 10% of freshwater tributaries and to protect the biological integrity of existing healthy watersheds. To achieve these goals, resource managers need to better understand which stressors are most likely driving biological impairment. Our study addressed this knowledge gap through two approaches: 1) reviewing and synthesizing published multi-stressor studies, and 2) examining 303(d) listed impairments linked to biological impairment as identified by jurisdiction regulatory agencies (the states within the watershed and the District of Columbia). Results identified geomorphology (i.e., physical habitat), salinity, and toxic contaminants as important for explaining variability in benthic community metrics in the literature review. Geomorphology (i.e., physical habitat and sediment), salinity, and nutrients were the most reported stressors in the jurisdictional impairment analysis. Salinity is likely a major stressor in urban and mining settings, whereas geomorphology was commonly reported in agricultural settings. Toxic contaminants, such as pesticides, were rarely measured; more research is needed to quantify the extent of their effects in the region. Flow alteration was also highlighted as an important urban stressor in the literature review but was rarely measured in the literature or reported by jurisdictions as a cause of impairment. These results can be used to prioritize stressor monitoring by managers, and to improve stressor identification methods for identifying causes of biological impairment.