Eucalyptus are a widely used short‐rotation species for timber production in South China. There are growing concerns regarding the influences of successive planting of Eucalyptus urophylla on microorganisms and soil. The aim of this work was to determine the effects of the successive planting of E. urophylla on soil characteristics. Five different stands, including 5‐, 10‐, 15‐, and 18‐year‐old E. urophylla plantations (abbreviated as 5yr, 10yr, 15yr, and 18yr, respectively) and one native evergreen broadleaf forest as a 0‐year‐old E. urophylla plantation (abbreviated as CK) were used. All of measured chemical indicators, microbial biomass, and enzyme activities were significantly lower in E. urophylla plantations compared with CK stand. The microbial biomass and enzyme activities significantly decreased in 5yr stand, significantly increased in 10yr stand, and subsequently declined in 15yr and 18yr stands. The relative abundance of Ascomycota in 15yr (77.10%) and 18yr (72.71%) plantations was significantly higher than in other stands. Basidiomycota relative abundance in the E. urophylla plantations was significantly lower than in the CK stand. In addition, the bacterial diversity was significantly decreased in 15yr and 18yr stands, whereas the fungal diversity was the lowest in 5yr stand and significantly increased in 10yr, 15yr, and 18yr stands. Furthermore, structural equation modelling showed that successive planting of E. urophylla decreased the soil bacterial community diversity, microbial biomass, and enzyme activity but increased the fungal community diversity. Thus, successive planting of E. urophylla negatively influenced the soil bacterial diversity, microbial biomass, and enzyme activity and positively influenced soil fungal diversity.