Changes in aquatic environments are potentially large, and effects on biota are highly variable. In Santa Catarina State, Brazil, the main sources of pollution in headwaters are wastewater, pesticides, and animal waste, which contain chemical and organic additives, and there is little knowledge about their effects on aquatic organisms. This study was aimed at investigating the possible genotoxic effects of the waters of the Engano River through the micronucleus (MN) test in Astyanax bimaculatus erythrocytes. Samplings were performed at two sites on the river, and there were six temporal repetitions with collection of blood from six individuals per site/repetition. For the negative-control treatment, we used fish from organic culture; cyclophosphamide was inoculated to constitute the positive-control treatment. MN was obtained in 3,000 erythrocytes/individual. Kruskal-Wallis analysis of variance, Scott-Knott test, and Spearman correlation were employed for data analysis. We found 124 MN in fish from the river, with 70 and 54 MN, respectively, for each site and 16 and 59 MN in negative and positive controls, respectively. The results showed a statistical difference for the formation of MN between river fish and fish in the positive-control treatment. Considering the average MN, we found the formation of three distinct groups: (1) fish from site no. 1 and those in the negative-control group, (2) fish from site no. 2, and (3) fish in the positive-control group. The greater values of chromosomal damage were found during periods of lower rainfall. The test used indicated the presence in the water of substances likely to cause clastogenic and aneugenic effects.