During 2014, the U.S. Geological Survey (USGS) National Water-Quality Assessment(NAWQA) project assessed stream quality in 75 streams across an urban disturbance gradient within the Piedmont ecoregion of southeastern United States. Our objectives were to identify primary instream stressors affecting algal, macroinvertebrate and fish assemblages in wadeable streams. Biotic communities were surveyed once at each site, and various instream stressors were measured during a 4-week index period preceding the ecological sampling. The measured stressors included nutrients; contaminants in water, passive samplers, and sediment; instream habitat; and flow variability. All nine boosted regression tree models – three for each of algae, invertebrates, and fish – had cross-validation R² (CV R²) values of 0.41 or above, and an invertebrate model had the highest CV R² of 0.65. At least one contaminant metric was important in every model, and minimum daytime dissolved oxygen (DO), nutrients, and flow alteration were important explanatory variables in many of the models. Physical habitat metrics such as sediment substrate were only moderately important. Flow alteration metrics were useful factors in eight of the nine models. Total phosphorus, acetanilide herbicides and flow (time since last peak) were important in all three algal models, whereas insecticide metrics (especially those representing fipronil and imidacloprid) were dominant in the invertebrate models. DO values below approximately 7 mg/L corresponded to a strong decrease in sensitive taxa or an increase in tolerant taxa. DO also showed strong interactions with other variables, particularly contaminants and sediment, where the combined effect of low DO and elevated contaminants increased the impact on the biota more than each variable individually. Contaminants and flow alteration were strongly correlated to urbanization, indicating the importance of urbanization to ecological stream condition in the region.