Nitrogen and carbon stable isotopes (δ N, δ C) are commonly used to understand mercury (Hg) bioaccumulation and biomagnification in freshwater food webs. Though sulfur isotopes (δ S) can distinguish between energy sources from the water column (aqueous sulfate) and from sediments to freshwater organisms, little is known about whether δ S can help interpret variable Hg concentrations in aquatic species or food webs. Seven acidic lakes in Kejimkujik National Park (Nova Scotia, Canada) were sampled for biota, water, and sediments in 2009 and 2010. Fishes, zooplankton, and macroinvertebrates were analyzed for δ S, δ N, δ C, and Hg (methyl Hg in invertebrates, total Hg in fishes); aqueous sulfate and profundal sediments were analyzed for δ S. Within lakes, mean δ S values in sediments and sulfate differed between 0.53‰ and 1.98‰, limiting their use as tracers of energy sources to the food webs. However, log-Hg and δ S values were negatively related (slopes -0.14 to -0.35, R 0.20-0.39, p < 0.001-0.01) through each food web, and slopes were significantly different among lakes (analysis of covariance, lake × δ S interaction term p = 0.04). Despite these relationships, multiple regression analyses within each taxon showed that biotic Hg concentrations were generally better predicted by δ N and/or δ C. The results indicate that δ S values are predictive of Hg concentrations in these food webs, although the mechanisms underlying these relationships warrant further study. Environ Toxicol Chem 2017;36:661-670. © 2016 SETAC.