Mesotrione is one of the new herbicides that have emerged as an alternative after the ban of atrazine in the European Union. To our knowledge, any work using genetic or biochemical biomarkers was performed in any kind of fish evaluating the toxicity of this compound. The impact of acute (96 h) exposure to environmentally relevant mesotrione concentrations (1.8, 7, 30, 115 e 460 μg L−1) were evaluated on the liver of Oreochorimis niloticus and Geophagus brasiliensis by assessing the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S- transferase (GST), the levels of reduced glutathione (GSH), carbonyl assays (PCO) and lipid peroxide (LPO) as well as the DNA damage to erithrocytes, liver and gills through the comet assay. We observed an increase in the concentration of GSH and the GPx activity in O. niloticus, and the GST and SOD activity in G. brasiliensis. We found significant increase in DNA damage in all tissues in both species. The results indicated that the acute exposure to mesotrione can induce oxidative stress and DNA damage in both species.