This study evaluated the relations between algal, invertebrate, and fish assemblages and physical environmental characteristics of streams at the reach, segment, and watershed scale in agricultural settings in the Midwest. The 86 stream sites selected for study were in predominantly agricultural watersheds sampled as part of the U.S. Geological Survey's National Water-Quality Assessment Program. Species abundance and over 130 biological metrics were used to determine which aspects of the assemblages were most sensitive to change at the three spatial scales. Digital orthophotograph-based riparian land use/land cover was used for analyses of riparian conditions at the reach and segment scales. The percentage area of different land-use/land-cover types was also determined for each watershed. Out of over 230 environmental characteristics examined, those that best explained variation in the biotic assemblages at each spatial scale include the following: 1) reach: bank vegetative cover, fine silty substrate, and open canopy angle; 2) segment: woody vegetation and cropland in the 250-m riparian buffer, and average length of undisturbed buffer; and 3) watershed: land use/land cover (both total forested and row crop), low-permeability soils, slope, drainage area, and latitude. All three biological assemblages, especially fish, correlated more with land use/land cover and other physical characteristics at the watershed scale than at the reach or segment scales. This study identifies biotic measures that can be used to evaluate potential improvements resulting from agricultural best-management practices and other conservation efforts, as well as evaluate potential impairment from urban development or other disturbances.