We examined mercury concentrations in three fish assemblages to estimate biomagnification rates in the Iténez main river, affected by anthropogenic activities, and two unperturbed rivers from the Iténez basin, Bolivian Amazon. Rivers presented low to moderate water mercury concentrations (from 1.25 ng L-1 to 2.96 ng L-1) and natural differences in terms of sediment load. Mercury biomagnification rates were confronted to trophic structure depicted by carbon and nitrogen stable isotopes composition (δ15N; δ13C) of primary trophic sources, invertebrates and fishes. Results showed a slight fish contamination in the Iténez River compared to the unperturbed rivers, with higher mercury concentrations in piscivore species (0.15 μg g-1 vs. 0.11 μg g-1 in the unperturbed rivers) and a higher biomagnification rate. Trophic structure analysis showed that the higher biomagnification rate in the Iténez River could not be attributed to a longer food chain. Nevertheless, it revealed for the Iténez River a higher contribution of periphyton to the diet of the primary consumers fish species; and more negative δ13C values for primary trophic sources, invertebrates and fishes that could indicate a higher contribution of methanotrophic bacteria. These two factors may enhance methylation and methyl mercury transfer in the food web and thus, alternatively or complementarily to the impact of the anthropogenic activities, may explain mercury differences observed in fishes from the Iténez River in comparison to the two other rivers.