Human exposure to mercury (Hg) remains a global public health concern, particularly in rural and agricultural communities, where biomonitoring data are scarce. This study assessed total Hg concentrations in blood and urine samples from 290 farmers living in 19 communities in the southwestern region of Espírito Santo State, Brazil. Additionally, the stable isotopic composition of carbon (δ13C) and nitrogen (δ15N) was analyzed in blood to investigate associations with dietary patterns. We also evaluated associations with demographic and lifestyle factors, including age, sex, smoking status, and alcohol consumption. Results revealed elevated blood Hg concentrations with substantial spatial variability across localities. Stable isotope analysis indicated a positive association between δ15N and blood Hg, suggesting that diets richer in animal protein may contribute to increased exposure. A negative association with δ13C further suggested that greater consumption of C3-derived staple foods may be linked to higher Hg exposure, possibly due to environmental contamination of local food sources or specific dietary pathways. In contrast, urinary Hg concentrations showed negative associations with age and body mass index, possibly reflecting physiological influences on excretion efficiency. While mean blood Hg concentrations were higher among smokers and frequent alcohol consumers, these differences were not statistically significant. The reference values (95th percentiles; P95) for blood and urinary Hg were 14.0 and 0.78 μg/L, respectively, being blood values above typical levels observed in non-occupationally exposed populations. These findings underscore the need to expand human biomonitoring programs to include agricultural populations and to incorporate stable isotope analysis as a complementary tool for tracing dietary and environmental sources of Hg exposure.

