Fish are an important source of nutrients which may reduce risk of adverse health outcomes such as cardiovascular disease; however, fish may also contain significant amounts of environmental pollutants such as mercury, polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and perfluorinated compounds (PFCs, also called perfluoroalkyl compounds), which confer increased risk for adverse health effects. The Wisconsin Departments of Health Services and Natural Resources developed a survey instrument, along with a strategy to collect human biological samples to assess the risks and benefits associated with long-term fish consumption among older male anglers in Wisconsin. The target population was men aged 50 years and older, who fish Wisconsin waters and live in the state of Wisconsin. Participants provided blood and hair samples and completed a detailed (paper) questionnaire, which included questions on basic demographics, health status, location of catch and species of fish caught/eaten, consumption of locally caught and commercially purchased fish, and awareness and source of information for local and statewide consumption guidelines. Biological samples were used to assess levels of PCBs, PBDEs, PFCs (blood), and mercury (hair and blood). Quantile regression analysis was used to investigate the associations between biomarker levels and self-reported consumption of fish from the Great Lakes and other areas of concern, other locally caught fish, and commercially purchased fish (meals per year). Respondents had a median age of 60.5 (interquartile range: 56, 67) years. The median fish consumption was 54.5 meals per year, with most fish meals coming from locally-caught fish. Participants had somewhat higher mercury levels compared with the US general population, while levels of other contaminants were similar or lower. Multivariate regression models showed that consumption of fish from the Great Lakes and areas of concern was associated with higher levels of each of the contaminants with the exception of PBDEs, as was consumption of locally caught fish from other water bodies. All commercial fish consumption was also associated with both hair and blood mercury. When looking at specific PCB, PBDE and PFC analytes, consumption of fish from the Great Lakes and areas of concern was associated with higher levels of each of the individual PCB congeners examined, as well as higher levels of all of the PFCs examined, with the exception of PFHxS. Among the PFCs, locally caught fish from other water bodies was also associated with higher levels of each of the congeners examined except PFHxS. Finally, all commercial fish was associated with higher levels of PFHxS.