Gold mining is responsible for most Hg pollution in developing countries. The aims of this study were to assess the levels of total Hg (T-Hg) in human hair, fish, water, macrophyte, and sediment samples in the gold mining district of San Martin de Loba, Colombia, as well as to determine fish consumption-based risks for T-Hg ingestion. T-Hg levels were measured by electrothermal atomization and atomic absorption spectroscopy. The overall mean T-Hg level in hair for humans in the mining district of San Martin de Loba was 2.12 μg/g, whereas for the reference site, Chimichagua, Cesar, it was 0.58 μg/g. Mean T-Hg levels were not different when considered within localities belonging to the mining district but differed when the comparison included Chimichagua. T-Hg levels in examined locations were weakly but significantly associated with age and height, as well as with fish consumption, except in San Martin de Loba. High T-Hg concentrations in fish were detected in Pseudoplatystoma magdaleniatum, Caquetaia kraussii, Ageneiosus pardalis, Cyrtocharax magdalenae, and Triportheus magdalenae, whereas the lowest appeared in Prochilodus magdalenae and Hemiancistrus wilsoni. In terms of Hg exposure due to fish consumption, only these last two species offer some guarantee of low risk for Hg-related health problems. Water, floating macrophytes, and sediments from effluents near mining sites also had high Hg values. In mines of San Martin de Loba and Hatillo de Loba, for instance, the geoaccumulation index (I(geo)) for sediments reached values greater than 6, indicating extreme pollution. In short, these data support the presence of a high Hg-polluted environment in this mining district, with direct risk for deleterious effects on the health of the mining communities.