Despite fresh and pyrogenic organic matter have been widely used as amendments to improve soil organic carbon (SOC) storage, mineralization that links to C quality and soil temperature, microbial community composition and enzyme activity remain poorly understood. This study aims to explore the effects of amendments (bamboo leaves and its biochar) and incubation temperature on mineralization, and disentangle the relationships of SOC mineralization with chemical composition of SOC, labile organic C, microbial community composition, and activities of enzymes in a subtropical bamboo forest soil. Results showed that cumulative soil CO2 emissions ranked as bamboo leaf (Leaf) > bamboo leaf biochar (Biochar) > Control, regardless of the incubation temperature. Compared to the control, the Leaf treatment markedly increased, whereas the Biochar treatment decreased, the temperature sensitivity of SOC mineralization (P < 0.05). The cumulative soil CO2 emission was positively correlated (P < 0.05) with water-soluble organic C (WSOC), microbial biomass C (MBC), O-alkyl C and alkyl C contents, and activities of ß-glucosidase and dehydrogenase, but negatively correlated (P < 0.01) with aromatic C content, regardless of the incubation temperature. This indicated that the lower SOC mineralization rate and lower temperature sensitivity in the Biochar (cf. Leaf) treatment were intimately associated with the lower WSOC, MBC, O-alkyl C content, and ß-glucosidase and dehydrogenase activities, and higher aromatic C content in the Biochar. The high relative abundance of bacteria relating SOC mineralization included Rhizobiales, Sphingobacteriales and JG30-KF-AS9, whereas that of fungi included Eurotiales, Sordariales, Agaricales and Helotiales. Our results revealed that the application of pyrogenic organic matter, as compared to the application of fresh organic matter, can reduce SOC mineralization and its temperature sensitivity in a subtropical forest soil by limiting the availability of C and microbial activity, and thus has a great potential for maintaining soil carbon stock in subtropical forest ecosystems.