Objectives: The aim of the research is to examine the values of zinc concentrations in serum, urine and hair in the population of eastern Croatia and to relate the obtained results to age, sex, previous diseases, professional exposure, place of residence and water consumption. Study design: A cross-sectional study was conducted. Participants and methods: The research was conducted on 601 randomly selected respondents from the patient databases of family medicine offices. The samples were collected in 2010 in Vladislavci, Dalje, Čepin, Našice, Osijek, Vukovar, Vinkovci, Slavonski Brod and in 2020 in Virovitica. Respondents filled out a standardized questionnaire consisting of 14 questions, which included basic sociodemographic indicators and habits. The collected samples were analyzed by the ICP-MS method. Results: There is a difference in zinc concentrations with regard to the sex and age of the subjects. Professionally exposed subjects have significantly higher concentrations of zinc in their hair. There are no differences in zinc concentrations in biological samples between subjects with and those without previous diseases. Higher concentrations of zinc were found in serum in subjects who consumed water from the city water supply, while higher concentrations in hair were found in those consuming local water supply. Higher concentrations of zinc in serum were found in subjects from Vukovar, Vinkovci and Virovitica, urine in subjects from Virovitica and zinc levels in hair were higher in Dalj, Čepin, Našice and Osijek. Conclusion: Despite the fact that zinc is an essential element that is important for human health, zinc is also a metal that, in elevated concentrations, is an indicator of anthropogenic pollution. By comparing the reference values of zinc in biological samples and the values obtained by biomonitoring, it seems that there is no significant contamination of the population of eastern Croatia with zinc.