The streams and rivers of the Upper Paraná River Basin have been seriously affected by impacts of high population density around the basin area. Fishes are widely used as models to assess the health of aquatic ecosystems, being considered as bioindicators of environmental pollution. In this context, our objective was to assess the potential genotoxic and mutagenic effects of the polluted water in three native fish species (Astyanax lacustris, Hypostomus ancistroides, and Rhamdia quelen) from Tarumã Microbasin, Upper Paraná River, Brazil. We also investigated the concentration of metals in water and in fish muscle to verify bioavailability and bioaccumulation of metals. For both less impacted sites (LI) and impacted sites (IMP) of the microbasin, the concentrations of metals were above the maximum limit allowed by Brazilian legislation (Resolution CONAMA 357/2005), except for Pb, total Cr, and Cu at LI sites and total Cr at IMP sites. A. lacustris showed a higher frequency of micronuclei (MN) at IMP sites compared with LI sites (p < 0.0001). We found no significant differences in MN frequency between site classes for H. ancistroides and R. quelen (p > 0.05). There were no significant differences between site classes regarding to nuclear abnormalities in erythrocytes frequencies (p > 0.05). A. lacustris from IMP sites had higher concentrations of Pb, Cu, Fe, Zn, and Ni in muscle tissue (p < 0.05), whereas H. ancistroides from IMP sites had higher concentration of Cr, Cu, and Ni (p < 0.0001) and R. quelen showed higher concentration of Cd, Fe, and Ni at these sites (p < 0.0001). So, the chosen biomarkers are able to identify the environmental risk of the water pollution.