This study was conducted on the continental shelf surrounding a large metropolitan region on the coast of São Paulo State, Southeast Brazil. This region harbours a large industrial plant and the largest port in Latin America, both of which release pollutants into the Santos−São Vicente estuarine system. High levels of Hg have been reported in sediments and fish from the estuaries and Santos Bay; however, data for the biota in offshore waters are scarce, and the biomagnification of Hg across the food web here has never been assessed. In this study, the trophic structure of the Santos shelf was addressed through the carbon and nitrogen stable isotope compositions of different species across a trophic gradient. We determined the total Hg levels (THg, dry weight) of invertebrates and fish to estimate the rate of biomagnification of this metal in the benthic and pelagic food webs. The lowest mean THg levels were found in zooplankton (0.006 µg g ). Overall, the Hg concentration in fish was below the recommended limit for human consumption. The THg and δ 15 N were positively correlated in both food webs; however, the rate of biomagnification was higher and the basal Hg was lower in the pelagic food web. These differences may be related to the differing bioavailability of mercury in water and sediment, the higher diversity of prey and more complex feeding interactions in the benthic food web, and metabolic differences among different taxa.