The decay of community similarity with distance (distance decay) is reported for many taxa in a variety of geographic settings. However, the importance of scale, distance measure, ecoregions, and ecological transition zones to distance decay has not been thoroughly examined. The goal of our study was to test the effects of these factors on distance decay in two freshwater assemblages (benthic macroinvertebrates and fish) with differing dispersal abilities in small streams within the Patuxent River basin, Maryland, USA. The Patuxent basin contains a geologic Fall Line, an ecological transition zone separating the two main regions of the basin. For both assemblages, we examined distance decay in similarity at several extents: entire Patuxent, Piedmont sub-region, and Plains sub-region using both linear geographic and stream network distances. Decay patterns were observed across all extents and distances. At the Patuxent extent decay rates differed between linear and stream distance only for macroinvertebrates (linear>stream); with both distance measures, similarity in fish decayed faster than similarity in macroinvertebrates. Within the Plains, decay rates for macroinvertebrates were lower than at the Patuxent for both distance measures; no difference in decay rates for this assemblage were detected in the Piedmont. Decay rates of similarity for fish only differed (lower) from rates at the Patuxent when examined at the Piedmont extent with stream distance. Similarity for the subset of sites that were located in separate ecoregions decayed at a slower rate than similarity for the entire data set only for macroinvertebrates with linear distance, suggesting a weak effect of the transition zone on distance decay. Together, these results suggest multiple factors contribute to the distance decay pattern and therefore regional diversity patterns, suggesting conjoint examination of these factors will further our understanding of the mechanisms governing regional diversity patterns.