Mercury (Hg) is a heavy metal that, once in the environment, is bioaccumulated and biomagnified through food chain impacting ecosystems. The aim of this study was to evaluate total Hg (T-Hg) concentrations in individuals along Cauca and Magdalena Rivers in Colombia, where most gold mining activities take place. A total of 1,328 hair samples were collected and analyzed for T-Hg using atomic absorption spectroscopy. T-Hg concentrations ranged from 0.01 to 20.14 μg/g. Greatest levels were detected in La Raya (5.27 ± 0.32 μg/g), Achi (2.44 ± 0.22 μg/g), and Montecristo (2.20 ± 0.20 μg/g), places that are located near gold mines. Concentrations decreased with the distance from main mining areas. Only 0.75% of the individuals had T-Hg levels above 10 μg/g. Men had significantly higher T-Hg levels than women, and correlation analysis revealed moderately weak but significant relationships between T-Hg and weight (R = 0.111, P < 0.001), stature (R = 0.111, P < 0.001), and age (R = 0.073, P = 0.007). However, T-Hg concentrations did not vary according to fish consumption frequency. Subjective health survey showed no Hg-related signs or symptoms within studied sample. However, studies are necessary to detect neurological damage linked to the metal. Changing technologies to Hg-free mining, monitoring, and educational programs are necessary to protect health of people living near Colombian rivers.