The Atrato River basin was protected by Colombian law due to anthropogenic impacts, mainly from illegal gold mining, which triggered a critical environmental health problem. In this study we quantified mercury (Hg), methylmercury (MeHg) and arsenic (As) concentrations in aquatic environmental matrices, and explored for the first-time nuclear degenerations in fish from the Atrato River. The median concentrations (μg/kg) for T-Hg, MeHg and As in fish were 195.0, 175.5, and 30.0; in sediments (μg/kg) 165.5, 13.8 and 3.1; and in water (ng/L), 154.7 for T-Hg and 2.1 for As. A 38% and 10% of the fish exceeded the WHO limit for the protection of populations at risk (200 μg Hg/kg) and for human consumption (500 μg Hg/kg); while As concentrations were below the international standard (1000 μg/kg) in all fish. The percentage of MeHg was 89.7% and the highest accumulation was observed in carnivorous fish (336.3 ± 245.6 μg/kg, p < 0.05) of high consumption, indicating risk to human health. In water, T-Hg concentrations exceeded the threshold effect value of 12 ng/L, whereas As concentrations were below the threshold of 10,000 ng/L, established by USEPA. On the contrary, 33% of the sediments exceeded the quality standard of 200 μg/kg for Hg. We found that Prochilodus magdalenae was the species with the highest susceptibility to nuclear alterations in its order, nuclear bud (CNB, 3.7 ± 5.4%), micronuclei (MN, 1.6 ± 2.5%) and binucleated cells (BC, 1.6 ± 2.3%). These results indicate that the species appears to be a good predictor of genotoxicity in the Atrato River. Fulton's condition factor (K) indicated that 31.7% of the fishes had poor growth condition, suggesting that the Atrato River basin needs to be monitored and restored in accordance with the agreements reached in the Minamata Convention on Mercury.