In Colombia, the inhabitants of the Mojana region have historically been subjected to high levels of environmental and occupational exposure to mercury; however, there are few robust data on the magnitude of this exposure and associated factors. This study aimed to describe the levels of mercury in the workers and inhabitants in this region, and to identify the main sociodemographic and occupational factors that are associated with this exposure. A cross-sectional study was conducted, in which mercury levels were determined in biological samples (blood, urine, hair) from 1119 people in the Mojana region. A questionnaire was also administered, which was adapted from the Global Mercury Assessment. Linear regression models were adjusted for the natural logarithm of mercury levels in blood, urine, and hair, using the factors that were explored as independent variables. The study reports high mercury levels in 35.0% of blood samples (95% CI 31.9–38.1%), 28.8% (95% CI 24.9–32.8%) of urine samples, and 56.3% (95% CI 53.1–59.5%) of hair samples. The reported source of water for consumption was associated with high levels of mercury (p-value < 0.05). We provide evidence of high levels of mercury exposure for the population in the Mojana region.