Soil microorganisms and extracellular enzymes play important roles in soil nutrient cycling. Currently, China has the second-largest area of eucalyptus plantations in the world. Information on the effects of eucalyptus age and species of trees on soil microbial biomass and enzyme activities, however, is limited. In this paper, the soil microbial biomass and enzyme activities were studied in eucalyptus plantations with different ages (1 and 5 years) and species of trees (. , and ) in South China. The results showed that both plantation age and eucalyptus species could affect the total microbial biomass and fungal biomass, whereas the bacterial biomass was affected only by plantation age. The fungal biomass and the fungi-to-bacteria ratio significantly increased along with increasing plantation age. Similarly, the plantation age and eucalyptus species significantly affected the enzyme activities associated with carbon cycling (β-xylosidase, β-d-glucuronidase, β-cellobiosidase and β-glucosidase). The activities of β-d-glucuronidase and β-glucosidase were significantly higher in the plantation. The enzymes involved in nitrogen (N-acetyl-glucosamidase) and sulfur (sulfatase) cycling were only affected by the eucalyptus plantation age and species, respectively. The results highlight the importance of the age and species of eucalyptus plantations on soil microbial activities.