The Veneto Region (Italy) experienced one of the heaviest contaminations by per- and polyfluoroalkyl substances (PFAS), pollutants of emerging concern due to their environmental persistence and bioaccumulation potential in animal tissues. Hence, there is a need to study their impact on freshwater fish inhabiting contaminated rivers, particularly at the level of the antioxidant system, since PFAS are known to cause an imbalance in reactive oxygen species (ROS) production, thereby increasing the risk of oxidative stress. This study examines the physiological responses triggered by chronic exposure to three distinct environmental concentrations of PFAS in the European chub (Squalius cephalus). The sites were classified as “control” (with a PFAS concentration < 5 ng/L), “low polluted” (5.64 ng/L) and “highly polluted” (582.6 ng/L). Biochemical and molecular analyses were performed on the kidney, one of the main organs for xenobiotic bioaccumulation. The catalase (CAT) and selenium-dependent glutathione peroxidases (Se-GPXs) expression was quantified at both active protein and mRNA transcript levels. Results confirm the activation of antioxidant defences against the risk of PFAS-induced oxidative stress. There is a differential induction in the biosynthesis of enzymes inside specific intracellular compartments: CAT in peroxisomes in the “low polluted” site and Se-GPXs in the cytoplasm in the “highly polluted” one. The gene gpx1 was the only isoform whose mRNA level corresponded to that of the active protein, suggesting the highest contribution to the biosynthesis of Se-GPXs at high PFAS concentrations. Conversely, gpx4 increased its transcription level in the “low polluted” site, which didn't match with an increase in protein content, leading us to hypothesise an involvement of specific cytoplasmic mRNA-protein complexes, called stress granules, acting in the temporary silencing of gpx4.