Understanding how biotic and abiotic processes influence community assembly is a fundamental aim in ecology. Although spatial scales at which communities are studied may affect the relative importance of such assembly processes, spatial influences on community assembly have not been thoroughly addressed. We tested how spatial scale affects inferences of habitat filtering and competitive exclusion assembly processes in darter (Percidae: Etheostomatinae) assemblages across four temperate stream systems. We predicted competitive exclusion would influence assembly in fine-scale assemblages, and habitat filtering would be more influential as spatial scale increased. Moreover, we assumed that habitat heterogeneity would increase with scale, and consequently alleviate direct competitive exclusion acting at finer scales. Using a framework that incorporated genetic relatedness, morphological traits, and habitat use among co-occurring darter species, we identified ecological and evolutionary patterns of structure, which allowed us to elucidate processes of assembly. Based on phylogenetic structure, assemblages showed an increase in habitat filtering (i.e., increased phylogenetic clustering) as we scaled up from fine to intermediate assemblages; however, we found mixed signals for habitat filtering and competitive exclusion at the broadest spatial scale. While habitat filtering was found to have an overall high relative importance during assembly, we also found influence of competitive exclusion processes based on limited morphological similarity among co-occurring species. Our results generally support an increased influence of habitat filtering processes in broader scale assemblages. Moreover, we suggest that habitat filtering and competitive exclusion processes act simultaneously during assembly, although the relative influence of each process may be spatial-scale dependent.