We explored land use, fish assemblage structure, and stream habitat associations in 20 catchments in Opequon Creek watershed, West Virginia. The purpose was to determine the relative importance of urban and agriculture land use on stream biotic integrity, and to evaluate the spatial scale (i.e., whole-catchment vs riparian buffer) at which land use effects were most pronounced. We found that index of biological integrity (IBI) scores were strongly associated with extent of urban land use in individual catchments. Sites that received ratings of poor or very poor based on IBI scores had > 7% of urban land use in their respective catchments. Habitat correlations suggested that urban land use disrupted flow regime, reduced water quality, and altered stream channels. In contrast, we found no meaningful relationship between agricultural land use and IBI at either whole-catchment or riparian scales despite strong correlations between percent agriculture and several important stream habitat measures, including nitrate concentrations, proportion of fine sediments in riffles, and the abundance of fish cover. We also found that variation in gradient (channel slope) influenced responses of fish assemblages to land use. Urban land use was more disruptive to biological integrity in catchments with steeper channel slopes. Based on comparisons of our results in the topographically diverse Opequon Creek watershed with results from watersheds in flatter terrains, we hypothesize that the potential for riparian forests to mitigate effects of deleterious land uses in upland portions of the watershed is inversely related to gradient.