Tree foliage sequesters gaseous elemental mercury (Hg) through stomatal uptake, when the foliage senesces and falls into the water, Hg from leaf litter can be released into the water and/or microbially methylated into a highly toxic form, methylmercury. The dominant groups of microbial communities that can methylate Hg during litter decomposition are, however, less certain. We conducted a microbial inhibition experiment to identify the primary methylators of leaf litter Hg during 28-day decomposition of two litter species of contrasting quality (pine and maple). We demonstrate that sulfate-reducing bacteria are the dominant microbial groups for Hg methylation during anoxic litter decomposition, and our study also indicates that methanogens may have a minor role in mediating Hg methylation during litter decomposition. Thus, aquatic environment with extensive litter accumulation and decomposition (e.g., wetlands, ponds, and river pools) can be hotspots of Hg methylation through sulfate-reduction and, to a lesser extent, methanogenesis.