Effective conservation of stream fishes and their habitats is complicated by the fact that human stressors alter the way in which natural factors such as stream size, catchment geology, and regional climate influence stream ecosystems. Consequently, efforts to assess the condition of stream fishes and their habitats must not only attempt to characterize the effects of human stressors but must account for the effects of natural influences as well. This study is an assessment of all stream fish habitats in the Mississippi River basin, USA. The basin supports over 400 stream fish species, drains a land area of 3.2 M km², and includes a myriad of human stressors such as intensive agriculture, urbanization, nutrient loading, and habitat fragmentation by dams and road/stream crossings. To effectively characterize types and levels of human stressors specifically impacting the basin’s stream fish species, our assessment approach first accounted for the influence of natural landscape conditions on species abundances with multiple steps, including stratifying our analyses by region and stream size and quantitatively modeling the influences of natural factors on stream fishes. We next quantified individual fish species responses to explicit human stressors for different measures of land use, fragmentation, and water quality, including summaries of measures in local vs. catchment extents. Results showed that many species had negative threshold responses to human stressors and that impacts varied by species, by region, and by the spatial extents in which stressors were summarized. Our spatially explicit results indicated the degree of stream reach impairment for specific stressor categories, for individual species, and for entire assemblages, all of which are types of information that can aid decision makers in achieving specific conservation goals in the region.