Microorganisms are vital in soil organic carbon (SOC) mineralization. The deposition of atmospheric nitrogen (N) and phosphorus (P), as well as leaf-litter addition, may affect SOC mineralization and microbial community structure by changing the availability of soil nutrients and carbon (C). In this study, we added leaf-litters labeled by 13C (Pinus massoniana and Michelia macclurei) and nutrients (ammonium chloride and monopotassium phosphate) alone and in combination to soils collected from a coniferous forest in subtropical China. We aimed to investigate the effect of leaf-litter and nutrient addition on SOC mineralization and soil microbial community. CO2 production was continuously measured during 120-day laboratory incubation, and CO2 sources were partitioned using 13C isotopic techniques. The addition of P. massoniana and M. macclurei leaf-litters increased SOC mineralization by 7.4% and 22.4%, respectively. N and P addition alone decreased soil respiration by 6.6% and 7.1%, respectively. Compared with P addition, N addition exerted a higher inhibitory effect on SOC mineralization induced by leaf-litter addition. Leaf-litter addition stimulated soil microbial activity and decreased the ratio of bacteria to fungi as a result of greater promotion on fungal growth. Moreover, 16:0 and 18:1u9c phospholipid fatty acids (PLFAs) had greater amount of 13C incorporation than other PLFAs, especially in nutrient-addition treatments. These results suggested that increased C input through leaf litter can stimulate SOC mineralization, whereas atmospheric N and P deposition can reduce this stimulatory effect and promote soil C storage in subtropical forests. Our results also illustrated that the use of 13C-labeled leaf litter coupled with 13C-PLFA profiling is a powerful tool for determining the microbial utilization of C.