The effects of human-induced stressors on riverine fish assemblages are still poorly understood, especially in species-poor assemblages such as those of temperate South American rivers. In this study we evaluated the effects of human-induced stressors on the taxonomic and functional facets of fish assemblages of two central-southern Chilean rivers: the Biobio River (flow regulated by multiple dams) and the Valdivia River (free-flowing). The study design considered reference condition, urban polluted, and urban-industrial polluted sites. To evaluate the effects of stressors on fish assemblages we assessed: (i) components of beta diversity; (ii) spatial and temporal patterns of fish structure using a multivariate approach; and (iii) functional diversity (specialization, originality, dispersion, and entropy) using linear models. We found a strong association between taxonomic and functional fish assemblage facets with a predominance of natural processes in the Valdivia River reflected in marked temporal dynamics. In contrast, the Biobio River showed a clear loss of association with seasonal pattern, and both taxonomic and functional facets appeared to respond significantly to pollution zones. Implementation of stricter environmental policies and integrated river basin management are instrumental for conserving species-poor fish assemblages in Chilean temperate rivers characterized by low functional traits redundancy.