•Fill the gap in the effect of stand density on microbial compositions.•The effect of stand density on fungi was higher than on bacteria.•Low-density plantations showed higher microbial diversity and richness.•Ascomycota and Basidiomycota were key species in response to stand density. Despite recent improvements in understanding the composition of microbial communities in forest soils, fundamental questions regarding how their dynamics and function vary with forest management strategies, such as stand density, and the factors that affect them are still unclear. In this study, the spectrophotometry and high-throughput sequencing technology were used to determine enzyme activities and microbial community composition in the topsoil (0–20 cm) of 35-year-old Chinese fir plantations with five stand densities. The results showed that stand density had significant effects on soil fungal community composition and diversity and the activity of catalase and sucrase. However, stand density had no significant effects on the bacterial communities, although the diversity of the bacterial communities varied with stand density. Overall, the increase in stand density reduced the activities of catalase, urease and surase and the Chao1 and Shannon diversity of bacterial and fungal communities. We found that positive correlations between enzyme activities and microbial diversity indices were significant. We also found that bacterial community compositions were significantly influenced by pH, alkali available N, available P and available K, and that fungal community composition was significantly influenced by pH, soil moisture, total N, alkali available N, available P and available K. Our results provide important insights into the effect of stand density on soil enzyme activities and microbial community composition, which is certainly significant for developing stand density regulation strategies to mitigate the effects of degradation of soil biological activities.