Diesel oil can be a source of contamination in aquatic environments, mainly as a result of spills. The effects of the water-soluble fraction of diesel (WSF) on Prochilodus vimboides were assessed. Fish were exposed to three different WSF dilutions for up to 96 h and were compared to a control group. Damages in the fragments of DNA were analyzed using the Comet assay. The presence of erytrocyts abnormalities was assessed by micronucleus test. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity and the accumulation of copper in gills were also analyzed. Fish exposed for 96 h had higher rates of damage than those exposed for 24 h. There was no significant difference regarding the presence of micronuclei between exposed and control fish and between 24-h and 96-h exposures. For AST, no significant difference was observed between samples collected at the two exposure times. Fish exposed to a 1:100 dilution of WSF showed higher activity of the enzyme ALT than the control fish after a 24-h exposure period. There was no bioaccumulation of copper in the gills. We conclude that the genotoxic effects of WSF in the cells are more evident in P. vimboides during an acute exposure.