Exposure to toxic trace elements, which include metals and metalloids, can induce adverse health effects, including life-threatening diseases. Conversely, essential trace elements are vital for bodily functions, yet their excessive (or inadequate) intake may pose health risks. Therefore, identifying levels and determinants of exposure to trace elements is crucial for safeguarding human health. The present study analyzed urinary concentrations of 14 trace elements (arsenic, cadmium, cobalt, chromium, copper, mercury, manganese, molybdenum, nickel, lead, antimony, selenium, thallium, and zinc) and their exposure determinants in 711 individuals, spanningfrom children to young adults from a Central European population from the Czech Republic. Multivariate linear regression and non-parametric Kruskal-Wallis ANOVA were used to investigate exposure determinants. Estimates of 95th percentile concentrations and confidence intervals were carried out to establish reference values(RV95). The study also assessed the percentage of population exceeding health-based guidance values (GVs) to gauge health risks. Young adults showed elevated toxic element concentrations, whereas children exhibited higher concentrations of essential elements. Mercury concentrations were associated with both dental amalgam filling count and seafood intake; arsenic concentrations were associated with seafood, rice, and mushroom consumption. Mushroom consumption also influenced lead concentrations. Sex differences were found for cadmium, zinc, nickel, and cobalt. Between 17.9% and 25% of the participants exceeded recommended GV for arsenic, while 2.4% to 2.8% exceeded GV for cadmium. Only one participant exceeded the GV for mercury, and none exceeded GVs for chromium and thallium. Essential trace elements' GVs were surpassed by 38% to 68.5% participants for zinc, 1.3% to 1.8% for molybdenum, and 0.2% to 0.3% for selenium. The present study examines trace element exposure in a Central European population from the Czech Republic, unveiling elevated exposure levels of toxic elements in young adults and essential elements in children. It elucidates key determinants of trace element exposure, including dietary and lifestyle indicators as well as dental amalgam fillings. Additionally, the study establishes novel reference values and acomparison with established health-based human biomonitoring guidance values, which are crucial for public health decision-making. This comprehensive biomonitoring study provides essential data to inform public health policies and interventions.