The environmental impact in natural populations of female Mediterranean killifish Aphanius fasciatus was monitored, in four sites of the Tunisian coast, by measuring metal contents (Cd, Cu, Zn) as well as levels of three oxidative stress biomarkers: Metallothioneins (MTs), Protein Sulfhydryl (PSH) and Thiobarbituric Acid-Reactive Substances (TBARS) in liver and gonad tissues. Site 2 appeared as the most polluted site, since it contains the highest concentrations of analyzed pollutants (Cu, Cd, Zn and PAHs) and presents the most important levels of metals in the liver and gonad tissues (P < 0.05). The significantly highest values of both Hepato-, Gonado-somatic indexes (HSI, GSI respectively) were noticed in Site 4 and Site 3. In liver tissues, the analysis of MTs and PSH levels revealed a significant difference between the studied sites (P < 0.05) while TBARS levels were similar. In gonad tissues, we noticed a significant difference for PSH contents between studied sites and the highest value was observed in female captured from the Site 4. Interestingly, these results suggest a possible sensitivity of liver and gonad tissue on the studied biomarkers content in A. fasciatus and will be helpful in pollution monitoring.