The Machado River is the main tributary of the Madeira River in the state of Rondônia, a region that has been impacted by deforestation, livestock, urban development, alluvial gold mining and urban and industrial effluents that likely contribute to the introduction of mercury (Hg) in the aquatic system. We aimed to determine the concentrations of total Hg (THg) in muscle and liver of Plagioscion squamosissimus, a main fishing resource in the region. Fish were sampled for two years at five sites, along a 90-km stretch of the middle Machado River. THg concentrations were analyzed in 64 muscle and 54 liver samples by cold vapor atomic absorption spectrophotometry (FIMS-400). THg levels were related with fish size and sex and with periods of the hydrological cycle. THg concentrations in both organs were positively and significantly related with fish body size. There was no significant variation in THg among periods of the hydrological cycle. Mean THg concentrations in muscle (1.09 ± 0.72 mg kg-1) and liver (1.28 ± 1.23 mg kg-1) were higher than the limit established by the WHO. Our results suggest that residual mercury from former alluvial gold extraction and lixiviation of mercury naturally occurring in the soil due to large-scale deforestaion is accumulating in the aquatic food chain in the Machado River. We conclude that it is necessary to monitor the levels of mercury in commercially important fish species in the region.