The impact of urbanization on stream ecosystems is linked by land cover changes to the alteration of the natural hydrology and subsequent physical disruption of stream biota and habitat. Seasonal floods are part of the natural disturbance regime of many streams, but urbanization increases their frequency and magnitude. This study evaluated the impact of hydrologic disturbance on fish and aquatic macroinvertebrates in 81 (56 urban/25 reference) Ohio streams. Hydrologic variables included annual and monthly 24-h rainfall maxima and computed annual peak discharge, with computation supported by GIS-based drainage area delineation and land cover characterization. Ohio biological criteria for fish and macroinvertebrates measured during the late spring and summer were negatively impacted by annual peak discharge in urban streams as compared to reference streams. Results support the application of stormwater best management practices as part of stream restoration efforts to mitigate urbanization impacts to fish and macroinvertebrates.