Anthropic activities often lead to the contamination of freshwater ecosystems by organic and inorganic pollutants with potential deleterious effects on wildlife health. However, some species such as the invasive mosquitofish (Gambusia holbrooki) can thrive in such polluted habitats, but the underpinning mechanisms are still unknown. The aim of this study was to characterize the physiological response of mosquitofish living along different pollution gradients in South of France. Eleven sites were selected according to various levels of pollutants in the water (pesticides, pharmaceuticals) and in mosquitofish tissue (PAHs, PBDEs, PCBs, organochlorines, metals). The level of the different pollutants varied among sites resulting in contrasted pollution gradients. The biological response of mosquitofish was measured using biomarkers of biotransformation, oxidative status, neurotoxicity and histopathological alteration in gills and liver. Muscle lipids, hepatosomatic condition, body condition and reproductive status were also measured. We used a Structural Equation Modelling (SEM) approach to characterize the direct and indirect effects of pollutants across biological levels. Results showed that high levels of POPs and metals affected biotransformation processes in both sexes, as well as non-enzymatic antioxidants level and resulted in gill histopathological alterations in females. In addition, pesticides increased the energetic demand reflected by reduced lipid storage in females and hepatosomatic condition in males. Interestingly, responses to pollution varied among sexes since females responded to a broader range of pollutant types than males. This study highlights some key traits underlying the tolerance to pollution of the mosquitofish, which could partly explain their invasive success in polluted ecosystems.