Metallic contamination is widespread, particularly in areas impacted by human activities. Human activities result in high loads of metals being discarded into the aquatic compartment, reinforcing the need to evaluate their toxic effects especially on exposed fish. The purpose of this study was to determine the toxic response (namely, antioxidant levels and lipoperoxidative damage) in both liver and gills of the freshwater fish species Gambusia holbrooki, exposed to lead and zinc. Fish were exposed for 28 days (chronic exposure) to ecologically relevant concentrations of the selected compounds. The following oxidative stress/damage biomarkers were evaluated: glutathione-S-transferases (GSTs), glutathione reductase (GR), and thiobarbituric acid reactive substances (TBARS). The results indicate that lead caused a significant oxidative response, with significant increase of the enzymatic antioxidant defense (GSTs activity in hepatic tissue, and GR activity in branchial tissue) of exposed organisms. On the other hand, zinc caused a significant inhibition of G. holbrooki hepatic GR, a biological response that may be related to the antioxidant activity exhibited by this metal. The obtained results are of high importance, especially if one considers that the obtained toxic responses occurred at low, albeit ecologically relevant, levels of exposure.