The aim of this study was to assess the potential genotoxic effects of produced water (PW) from an Italian on-shore oil plant. Produced water is a complex mixture containing residual hydrocarbons, trace elements, naturally occurring radioactive material and potentially toxic treatment chemicals such as biocides, dispersants, detergents and scale inhibitors used in oil production. The test organism, mosquito fish (Gambusia affinis), was divided into male and female groups and exposed for 8 days in the laboratory to 50% concentrations of different produced waters: PW before treatment and after settling treatment. The fish were also exposed to lower concentrations (10%) of the same PW for 30 days. DNA damage was evaluated in erythrocytes by single cell gel electrophoresis (Comet assay) and micronucleus test, while an oxidative stress biomarker, was assessed. Polycyclic aromatic hydrocarbons (PAH) metabolites in bile were also evaluated. A higher sensitivity in biomarker responses was found in females in comparison to males. An increase in DNA strand breaks was observed in both genders after 30 days exposure and a statistically significant increase of micronucleated cells was found in females after 8 days exposure. A positive correlation between presence of micronucleated cells and PAH metabolites in bile was also observed.