Regionally scaled assessments of hydrologic alteration for small streams and its effects on freshwater taxa are often inhibited by a low number of stream gages. To overcome this limitation, we paired modeled estimates of hydrologic alteration to a benthic macroinvertebrate index of biotic integrity data for 4522 stream reaches across the Chesapeake Bay watershed. Using separate random-forest models, we predicted flow status (inflated, diminished, or indeterminant) for 12 published hydrologic metrics (HMs) that characterize the main components of flow regimes. We used these models to predict each HM status for each stream reach in the watershed, and linked predictions to macroinvertebrate condition samples collected from streams with drainage areas less than 200 km2. Flow alteration was calculated as the number of HMs with inflated or diminished status and ranged from 0 (no HM inflated or diminished) to 12 (all 12 HMs inflated or diminished). When focused solely on the stream condition and flow-alteration relationship, degraded macroinvertebrate condition was, depending on the number of HMs used, 3.8–4.7 times more likely in a flow-altered site; this likelihood was over twofold higher in the urban-focused dataset (8.7–10.8), and was never significant in the agriculture-focused dataset. Logistic regression analysis using the entire dataset showed for every unit increase in flow-alteration intensity, the odds of a degraded condition increased 3.7%. Our results provide an indication of whether altered streamflow is a possible driver of degraded biological conditions, information that could help managers prioritize management actions and lead to more effective restoration efforts.