To examine down-estuary effects and how differences in food webs along a salinity gradient might influence mercury (Hg) biomagnification, we conducted a study from 2010 to 2015 in an estuary with a known biological hotspot at its headwaters. Over 907 samples of biota, representing 92 different taxa of fish and invertebrates, seston and sediments were collected from the upper, middle and lower reach for Hg determination and for stable nitrogen and carbon isotope analyses. Trophic magnification slopes (TMS; log Hg versus δ15N), as a measure of biomagnification efficiency, ranged from 0.23 to 0.241 but did not differ statistically among reaches. Hg concentrations were consistently highest, ranging as high as 4.9 mg/kg in top predatory fish, in the upper-reach of the estuary where basal Hg entering the food web was also highest, as evidenced by methylmercury concentrations in suspension feeders. Top predatory fish at the mouth of the estuary contained relatively low [THg], likely due to lower basal Hg. This was nonetheless surprising given the potential for down-estuary biotransport.