Methylmercury (MeHg) is a neurotoxin, mainly derived from microbial mercury methylation in natural aquatic environments, and poses threats to human health. Polar regions and paddy soils are potential hotspots of mercury methylation and represent environmental settings that are susceptible to natural and anthropogenic perturbations. The effects of changing environmental conditions on the methylating microorganisms and mercury speciation due to global climate change and farming practices aimed for sustainable agriculture were discussed for polar regions and paddy soils, respectively. To better understand and predict microbial mercury methylation in the changing environment, we synthesized current understanding of how to effectively identify active mercury methylators and assess the bioavailability of different mercury species for methylation. The application of biomarkers based on the hgcAB genes have demonstrated the occurrence of potential mercury methylators, such as sulfate-reducing bacteria, iron-reducing bacteria, methanogen and syntrophs, in a diverse variety of microbial habitats. Advanced techniques, such as enriched stable isotope tracers, whole-cell biosensor and diffusive gradient thin film (DGT) have shown great promises in quantitatively assessing mercury availability to microbial methylators. Improved understanding of the complex structure of microbial communities consisting mercury methylators and non-methylators, chemical speciation of inorganic mercury under geochemically relevant conditions, and the pathway of cellular mercury uptake will undoubtedly facilitate accurate assessment and prediction of in situ microbial mercury methylation. Display omitted •Changing natural condition and human activity strongly affect mercury methylation.•Microorganisms possessing hgcAB genes are present in diverse habitats.•Mercury isotopes, whole-cell biosensor and DGT can assess mercury bioavailability.