Human exposure to per- and poly-fluoroalkyl substances (PFAS) occurs mainly through two pathways, inhalation and ingestion. Dietary exposure to persistent organic pollutants (POPs), including PFAS, is driven mainly by the consumption of foods of marine or terrestrial animal origin. Therefore, the intake of fish from waters in populated or polluted areas may be a source of human exposure to PFAS. The overall aim of the study was to estimate human exposure to PFAS through the consumption of fish caught in Swedish waters. Analyses of extractable organofluorine (EOF) were performed to serve as an important metric alongside target analysis to better understand the total amount of PFAS in the human sera and fish samples. The serum samples represent individuals who have lived at some point about 5 km from the glass industry in Nybro and Emmaboda. For this study with the Glasbruket study population, the highest concentrations were found for PFOS followed by PFOA, PFNA and PFHxS (medians of 7.9, 1.9, 1.4 and 0.8 ng/mL). Also, the difference in this population between men/women and high/low fish consumers were also addressed. It was stated that there was a statistically significant difference in average Σ17 PFAS concentration between the male and female groups (p < 0.05, two-tailed test). However, the differences between the low and high consumer groups were not statistically significant even though the PFAS levels were higher in the high consumer group. The organofluorine mass balance analysis revealed that 80.1% (ranged from 68.3−93.7%) of the EOF in female samples could not be explained, whereas 57.3% (ranged from 0−99.4%) for the male group was of unidentified origin. Two methods were evaluated for PFAS and EOF analysis of fish muscle, namely, acetonitrile extraction and ion-pair extraction. The selected method, the ion-pair extraction, was performed on fish muscle samples. The fish species included perch (Perca fluviatilis), northern pike (Esox Lucius) and zander (Sander lucioperca) that were collected from seven different lakes in the vicinity of Nyro and Emmaboda. The sum of targeted PFAS (∑14PFAS) across all fish samples analysed ranged from 0.9 to 6.2 ng/g. Mostly, perfluoroalkyl carboxylic acids (PFCAs), precursors to PFCAs and novel PFAS were found in the fish samples. A large part of the EOF content cannot be identified with the targeted PFAS compounds. The average identified EOF fraction for all fish was 2.3% (ranging from 0.8 to 7.2%). For this study, 10 PFAS were found in both sera and fish samples. Therefore, freshwater fish consumption can be identified as one of the contributors to the PFAS concentrations in the Glasbruket population. The fish samples contained precursor compounds as well, that were not found in human serum. These precursor compounds can contribute to the concentrations of PFAAs in serum through biotransformation in the human body to perfluoroalkyl acids (PFAAs). Due to the widespread use of PFAS and their persistence in the environment, it is difficult to determine the relationship between the levels found in serum and fish. The Glasbruket population could be exposed to other sources besides fish.