Objectives: The aim of this study was to examine the values of arsenic concentration in serum, urine and hair in the population of eastern Croatia and its correlation to age, gender, occupational exposure, comorbidities, residence, habits and source of drinking water. Study design: A cross-sectional study was conducted. Participants and methods: The study included 596 adult respondents, randomly selected from medical records in family medicine clinics. The sampling area is the territory of eastern Croatia, which includes 5 counties and 9 settlements: Čepin, Dalj, Našice, Osijek, Vinkovci, Virovitica, Vladislavci, Vukovar and Slavonski Brod. Samples were collected during 2010 for all settlements except in Virovitica, where they were collected in 2020. Each respondent completed a standardized questionnaire consisting of 14 questions, which included basic sociodemographic indicators. Samples were analyzed by ICP-MS. Results: The results suggest that there is a significant difference in arsenic concentrations based on age and gender. The expected association between occupational exposure and previous comorbidities for elevated arsenic in biological samples was also determined. Significant values of arsenic were obtained from the respondents who consumed water from wells and city water supply compared to those who consumed bottled water and water from local water supply systems. In the settlements that were on the front line of defense during the war, higher concentrations of arsenic were recorded in biological samples. Conclusion: The occurrence of arsenic in the population of eastern Croatia is an important public health problem given the significant differences in arsenic concentrations. Chronic arsenic exposure leads to various health problems, which is why it is important to continue working on promoting biomonitoring and prevention of arsenic exposure.