The data on water pollution is scarce in developing countries, including Brazil. The water quality assessment is important implementing the monitoring and remediation programs to minimize the risk of hazardous substances in freshwaters. Thus, this study evaluated the surface water quality of a stretch of the Bois River (Brazil), based on the physicochemical, microbiological and ecotoxicological analyses conducted in 2017, using Standard Methods and fish embryo acute toxicity (FET) test with zebrafish (Danio rerio). The results indicated that the quality of water samples located close to the discharge of tannery effluents was most impaired. Total phosphorus, BOD, DO, ammoniacal nitrogen, and thermotolerant coliforms parameters in P4 were not in accordance with the standards of current Brazilian legislation. Iron, lead, and copper levels were higher than environmental standards. The physicochemical quality of water samples was lower in the dry season than the rainy season. All samples (P1, P3, and P5) in rainy and dry seasons did not induce significant acute toxicity for zebrafish early-life stage; however other trophic levels (algae and microcrustacean) should be investigated to gain a better understanding of the toxicity during water quality analysis. In conclusion, the physicochemical and microbiological changes in the water of the Bois River can affect aquatic organisms as well as humans when it is used for drinking or in agriculture.