The Funil reservoir receives a large amount of xenobiotics from the Paraíba do Sul River (PSR) from large number of industries and municipalities in the watershed. This study aimed to assess environmental quality along the longitudinal profile of the Paraíba do Sul river–Funil Reservoir system, by using biomarkers and bioindicators in a selected fish species. The raised hypothesis is that Funil reservoir acts as a filter for the xenobiotics of the PSR waters, improving river water quality downstream the dam. Two biomarkers, the ethoxyresorufin–O–deethylase activity (EROD), measured as fluorimetricly in S9 hepatic fraction, and the micronuclei frequency (MN), observed in erythrocytes of the cytoplasm, and three bioindicators, hepatosomatic index (HSI), gonadosomatic index (GSI) and condition factor (CF) were used in Pimelodus maculatus, a fish species widely distributed in the system. Four zones were searched through a longitudinal gradient: 1, river upstream from the reservoir; 2, upper reservoir; 3, lower reservoir; 4, river downstream of the reservoir. EROD activity and HSI and GSI had significant differences among the zones (P