Abstract: In order to explore the effects of cultivation duration of Ganoderma lucidum on the soil organic carbon fractions and microbial functional characteristics, Ganoderma lucidum forest plots with different cultivation durations (2, 4, and 6 years) were used as the object, and the primary broad-leaved forest was used as the control (CK). Then, the soil samples were collected from the 0–20 cm soil layer to determine the contents of different forms of soil organic carbon. The microbial metabolic activity and functional diversity were assessed by using Biolog EcoPlate™ technology. The results showed that Ganoderma lucidum cultivation under the forest had no significant effect on the soil total organic carbon (TOC) content. However, with the increase of cultivation duration, the contents of soil microbial biomass carbon (MBC) and water-soluble organic carbon (WSOC) exhibited an initial increase followed by stabilization. Specifically, the soil MBC and WSOC contents in the 2-year treatment were significantly higher than those in the other treatments (P＜0.05) , increasing by 29.99% and 28.67% compared to CK, respectively. The microbial metabolic activity, represented by the average well color development (AWCD) value, peaked in the 2-year treatment, with the AWCD values in CK and the 2-year treatment being significantly higher than those in the 4-year and 6-year treatments (P＜0.05). Moreover, the Shannon-Wiener diversity index, Simpson dominance index, McIntosh evenness index, and Pielou evenness index of soil microorganisms were also the highest in the 2-year treatment, indicating that the functional diversity of microbial communities was significantly enhanced during the initial cultivation stage. The principal component analysis showed that there were obvious differences in the utilization models of carbon sources by soil microorganisms, among which the carbohydrate carbon sources were the key factors to distinguish the microbial communities of G. lucidum cultivated and uncultivated soils. The correlation analysis further showed that the microbial functional diversity index was positively correlated with TOC, MBC and WSOC content. In conclusion, the cultivation of Ganoderma lucidum under the forest exhibited the soil ecological effect characterized by "short-term promotion and long-term inhibition". It was recommended to implement the intermittent cultivation or fallow systems to maintain the soil health and ensure the sustainability of understory planting patterns.