Anthropogenic disturbances are relatively common in freshwater systems; however, documenting and understanding disturbance-specific impacts on aquatic communities remains an area of needed focus. We examined the effects of wastewater treatment plant (WWTP) effluent on fish assemblage structure using 26 riffle sites positioned along two tributaries of the Niangua River, Missouri, USA (West Fork—WWTP effluent present, and East Fork—WWTP effluent absent). Comparisons of rarified species richness (i.e., using interpolation and extrapolation) indicated that α diversity was similar between forks, despite the West Fork having greater raw species richness. Multivariate analyses of fish assemblage structure (β diversity) revealed a significant difference between East and West Fork sites characterized by presence of Etheostoma flabellare (Fantail Darter) and greater abundances of Cottus bairdii (Mottled Sculpin), a heat-intolerant species, in East Fork sites. Analyses of abiotic site characteristics demonstrated that the West Fork generally contained smaller substrates and had warmer water temperatures during winter months, the latter of which may relate to WWTP effluent release in the upper West Fork. These results highlight significant structural differences between riffle fish assemblages in the presence and absence of WWTP effluent, suggesting effluent release may have localized and downstream impacts on stream community structure.