Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with bioaccumulation potential, particularly affecting aquatic ecosystems and human health also via fish consumption. There is therefore a need for reliable extraction methods and studies to accurately assess PFAS levels in fish, crucial for understanding bioaccumulation and potential toxicological effects on both fish and humans through consumption. This study investigated PFAS levels in freshwater fish from Swiss lakes, focusing on six common species: Coregonus wartmanni, Cyprinus carpio, Oncorhynchus mykiss, Perca fluviatilis, Salmo trutta, and Squalius cephalus. Utilizing an optimized QuEChERS extraction method, 15 PFAS were analyzed in 218 fish fillet samples using liquid chromatography-mass spectrometry (LC-MS/MS). The results were compared to EU regulations and EFSA guidelines for tolerable weekly intake (TWI), with a specific focus on correlations between fish size and PFAS concentration. Our findings reveal significant PFAS contamination, particularly in Perca fluviatilis with perfluorooctane sulfonic acid (PFOS) and perfluorohexane sulfonic acid (PFHxS) levels often exceeding EU safety limits. TWI, calculated for a person of 70 kg body weight and an intake of 200 g of fish fillet, is exceeded in 95% of Coregonus wartmanni, 100% of Squalius cephalus, and in 55%, 50%, and 36% of the specimens Oncorhynchus mykiss, Salmo trutta, and Perca fluviatilis respectively. Correlation analysis between PFAS concentration and fish size in 121 Salmo trutta specimens revealed significant positive correlations for perfluorobutane sulfonic acid (PFBS), perfluorodecanoic acid (PFDA), and perfluorohexane sulfonic acid (PFHxS), and a negative correlation for perfluoropentanoic acid (PFPeA). These results underscore the critical need for continuous monitoring and regulatory efforts to mitigate PFAS exposure risks to both ecosystems and human health.