Stream fish bioassessment methods assume that fish assemblages observed in sample sites reflect responses to local stressors, but fish assemblages are influenced by local factors as well as regional dispersal to and from connected streams. We hypothesized that fish movement to and from refugia and source populations in connected rivers (i.e., riverine dispersal) would weaken or decouple relations between fish community metrics and local environmental conditions. We compared fish-environment relations between streams that flow into large rivers (mainstem tributaries) and streams that lack riverine confluences (headwater tributaries) at multiple spatial grains using data from the USEPA's Environmental Monitoring and Assessment Program in the mid-Atlantic highlands, USA (n = 157 sites). Headwater and mainstem tributaries were not different in local environmental conditions, but showed important differences in fish metric responses to environmental quality gradients. Stream sites flowing into mainstem channels within 10 fluvial km showed consistently weaker relations to local environmental conditions than stream sites that lacked such mainstem connections. Moreover, these patterns diminished at longer distances from riverine confluences, consistent with the hypothesis of riverine dispersal. Our results suggest that (1) the precision of fish bioassessment metrics may be improved by calibrating scoring criteria based on the spatial position of sites within stream networks and (2) the spatial grain of fish bioassessment studies may be manipulated to suit objectives by including or excluding fishes exhibiting riverine dispersal.