Reducing Hg contamination in soil using eco-friendly approaches has attracted increasing attention in recent years. In this study, a novel multi-metal-resistant Hg-volatilizing fungus belonging to Lecythophora sp., DC-F1, was isolated from multi-metal-polluted mining-area soil, and its performance in reducing Hg bioavailability in soil when used in combination with biochar was investigated. The isolate displayed a minimum inhibitory concentration of 84.5mg·L−1 for Hg(II) and volatilized >86% of Hg(II) from LB liquid medium with an initial concentration of 7.0mg·L−1 within 16h. Hg(II) contents in soils and grown lettuce shoots decreased by 13.3–26.1% and 49.5–67.7%, respectively, with DC-F1 and/or biochar addition compared with a control over 56days of incubation. Moreover, treatment with both bioagents achieved the lowest Hg content in lettuce shoots. Hg presence and DC-F1 addition significantly decreased the number of fungal ITS gene copies in soils. High-throughput sequencing showed that the soil fungal community compositions were more largely influenced by DC-F1 addition than by biochar addition, with the proportion of Mortierella increasing and those of Penicillium and Thielavia decreasing with DC-F1 addition. Developing the coupling of Lecythophora sp. DC-F1 with biochar into a feasible approach for the recovery of Hg-contaminated soils is promising. Display omitted •A novel metal-resistant Hg(II)-volatilizing fungus, Lecythophora sp. DC-F1, was isolated.•DC-F1 and biochar both effectively reduced Hg(II) contents in soil and plants.•The soil with both bioagents exhibited the lowest Hg uptake in lettuce shoots.•Soil fungal abundance and community structure were influenced to a greater degree by DC-F1 addition than by biochar addition.