Methylmercury (MeHg) is a pervasive and ubiquitous environmental toxin that endangers human health worldwide. Certain anaerobic microorganisms, such as the sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 and iron-reducing bacterium Geobacter sulfurreducens PCA, are known to convert inorganic mercury to MeHg in natural environments. However, a small group of methanotrophs such as Methylosinus trichoorium OB3b has recently been shown to degrade MeHg by as yet an uncharacterized mechanism, in which methanobactin (MB) (a metal-binding thiol compound excreted by OB3b) plays an essential role. Here, we examined the opposite effects of MB on the biosynthesis of MeHg by D. desulfuricans ND132 and G. sulfurreducens PCA in laboratory culture studies since the thiol compound is also known to impact microbial methylation. Surprisingly, MB was found to greatly enhance both the rates and efficiency of MeHg production by ND132 and PCA. Compared to the cultures without MB, the methylation rate increased by 6-7 times, and > 90% of the added Hg(II) (25 nM) was converted to MeHg in 72 h by D. desulfuricans ND132. Mercury speciation analyses indicate that MB likely formed strong complexes with Hg(II) in solution and thus increased the bioavailability of Hg(II) for cell methylation. While the exact mechanism of MB-enhanced mercury methylation remains to be investigated, this study provides new considerations of complex environmental factors, such as the presence of novel exogenous thiol-containing ligands, in mercury methylation and demethylation in the environment.