Marine animals often accumulate various harmful substances through the foods they ingest. The bioaccumulation levels of these harmful substances are affected by the degrees of pollution in the food and of biomagnification; however, which of these sources is more important is not well-investigated for mercury (Hg) bioaccumulation. Here we addressed this issue in fishes that inhabit the waters around Minamata Bay, located off the west coast of Kyushu Island in Kumamoto Prefecture, Japan. The total Hg concentration (hereafter [THg]) and carbon and nitrogen stable isotope ratios (δ13C and δ15N) were analyzed in the muscle tissue of 10 fish species, of which more than five individuals were caught by gillnet. Except one species, each was separated into two trophic groups with respective lower and higher δ13C values ranging from −17‰ to −16‰ and −15‰ to −14‰, which suggested that the fishes depended more on either phytoplankton- and microphytobenthos-derived foods (i.e., pelagic and benthic trophic pathways), respectively. Linear mixed effects models showed that the Hg levels were significantly associated with both δ15N and the differences in the trophic groups. [THg] increased with δ15N (i.e., indicative of higher trophic levels), but the slopes did not differ between the two trophic groups. [THg] was significantly higher in the group with higher δ13C values than in those with lower δ13C values. The effect size from marginal R squared (R2) values showed that the variation in [THg] was strongly ascribed to the trophic group difference rather than δ15N. These results suggest that the substantial Hg bioaccumulation in the fishes of Minamata Bay is mainly an effect of ingesting the microphytobenthos-derived foods that contain Hg, and that the subsequent biomagnification is secondary.