The Dingtao M2 tomb, the largest and best-preserved imperial “Huangchangticou” tomb in China, holds great significance for its conservation. Currently, varying degrees of microbial degradation are occurring on the surfaces of the M2 tomb. This study aimed to determine the microbial diversity of the M2 tomb and its surrounding environment during July 2021 and August 2022. High-throughput metagenomic sequencing revealed that the dominant fungus on the surface of the tomb chamber was Dacrymyces stillatus (DTT1) in July 2021, which changed to Talaromyces pinophilus (DTT2) in August 2022. Enzymatic activities for cellulose and lignin degradation suggested that DTT1 has high levels of manganese peroxidase, lignin peroxidase, laccase, and cellulase. The wood of the tomb contained higher levels of Fe2+ and Ca2+, and experiments with different concentration gradients of these ions in the culture medium revealed that DTT1 exhibited greater activity of cellulose and lignin degradation in environments with higher concentrations of Fe2+ and Ca2+. DTT2 degraded both cellulose and lignin. Lastly, a laboratory plate inhibition experiment demonstrated that isothiazolinone fungicide had a significant fungicidal effect on these two dominant fungi. This study provides valuable data and a theoretical basis for the preservation of the M2 tomb and other wooden cultural relics.