In Latium (central Italy), arsenic concentrations exceeding the regulatory limit of 10 μg/L for drinking water are present in groundwater from a large area of volcanic origin. At least in part of the area, high arsenic concentrations have been detected also in soil and phytoavailable geogenic arsenic enters the food chain. As a result, local population may be exposed to inorganic arsenic via water and also through consumption of food with higher than background arsenic concentrations. A cross sectional study was conducted to assess inorganic arsenic exposure and metabolism in 269 residents of 27 municipalities in the provinces of Viterbo, Rome and Latina. Total arsenic in toenails and the sum of inorganic arsenic and methylated metabolites in urine, the latter determined by HPLC-ICP-MS, were used as biomarkers of inorganic arsenic exposure. All the subjects involved in the study provided samples of the water(s) used for drinking and cooking as well as detailed information on water use. To get an insight into dietary intake from locally-processed food, inorganic arsenic in bread samples collected in affected municipalities of the three provinces was determined and compared to background levels of samples from reference areas. 30% of the sample used bottled water or resorted to water treatment in order to lower the arsenic content <10 μg/L (Group 1), 51% of the sample drank bottled water and used tap water with an arsenic content exceeding 10 μg/L for cooking only (Group 2), 19% of the sample used tap water with an arsenic content exceeding 10 μg/L for both drinking and cooking (Group 3). Nail arsenic was higher for Group 2 and 3 compared to Group 1, whereas all groups had higher nail arsenic than the reference group. The sum of inorganic arsenic and related metabolites in urine was higher in Group 3 than in the other two groups, and higher in Group 2 compared to Group 1. White bread from the study area showed significantly higher inorganic arsenic levels compared to samples from reference areas. Use of toenail arsenic as biomarker of long-term exposure allowed to retrospectively reconstruct exposure irrespective of recent modifications due to changes in water use. In Group 3, urinary concentration of inorganic arsenic and metabolites exceeded the upper limit of the reference concentration range for the Italian population. Inter-individual variability of the efficiency of arsenic metabolism in the study population was substantial indicating that a subgroup of the population is more susceptible to the toxic effects of inorganic arsenic owing to a lower methylation capability.