The Madeira River is the largest tributary of the Amazon River Basin and one of the most impacted by artisanal gold-mining activities, deforestation for agricultural projects, and recent hydroelectric reservoirs. Total Hg (and methylmercury-MeHg) concentrations was determined in 3182 fish samples of 84 species from different trophic levels as a function of standard size. Species at the top of the trophic level (Piscivorous, Carnivorous) showed the highest mean total Hg concentrations (51–1242µg/kg), Planctivorous and Omnivorous species showed intermediate total Hg concentrations (26–494µg/kg), while Detritivorous and Herbivorous species showed the lowest range of mean total Hg concentrations (9–275µg/kg). Significant correlations between fish size (standard length) and total Hg concentrations were seen for Planctivorous (r=0.474, p=0.0001), Piscivorous (r=0.459, p=0.0001), Detritivorous (r=0.227, p=0.0001), Carnivorous (r=0.212, p=0.0001), and Herbivorous (r=0.156, p=0.01), but not for the Omnivorous species (r=−0.064, p=0.0685). Moreover, fish trophic levels influenced the ratio of MeHg to total Hg (ranged from 70% to 92%). When adjusted for standard body length, significant increases in Hg concentrations in the last 10 years were species specific. Spatial differences, albeit significant for some species, were not consistent with time trends for environmental contamination from past alluvial gold mining activities. Fish-Hg bioaccumulation is species specific but fish feeding strategies are the predominant influence in the fish-Hg bioaccumulation pattern. •We studied 84 species 3182 fish specimens from the Madeira River, Amazon Basin.•Fish total-Hg and MeHg were studied in relation to spatial and temporal changes.•Significant increases in Hg concentrations in the last 10 years were species specific.•Spatial differences were not consistent with time trends and gold mining legacy.