Eucalyptus is an important tree species for afforestation in tropical or subtropical regions and many countries around the world have used it in afforestation. In South China, millions of hectares of local forests have been converted into Eucalyptus plantations. However, due to soil nutrient limitation in many areas and a high demand of Eucalyptus for nutrients, this kind of change in land use may exhaust soil nutrients and lead to deterioration of the soil in quality. In order to explore impacts of the practice on soil quality, soil samples were collected from 9 pairs of Eucalyptus and Pinus plantations in Guangxi, China, for analysis of soil nutrients, soil microbial biomass and soil enzyme activities and paired-t test. Pearson correlation analysis was done to explore relationships between soil nutrients, soil microbial biomass and enzyme activities. Results show that the replacement significantly decreased soil total carbon, labile carbon pool (I) and (II), recalcitrant carbon pool, total nitrogen and alkalytic nitrogen, but significantly increased soil available phosphorus, which might be a result of fertilization in the Eucalyptus plantations low in phosphorus mobility in the soil. The replacement also significantly decreased soil microbial biomass carbon and nitrogen, and activities of phenol oxidase, peroxidase, protease, urease and acid phosphatase. The significantly lower soil carbon, nitrogen, microbial biomass and enzyme activities suggested that conversion of Pinus plantations to Eucalyptus ones significantly decreased soil quality. Correlation analysis shows that (1) soil total carbon and nitrogen were positively related with soil microbial biomass carbon, microbial biomass nitrogen, cellobiosidase, peroxidase, protease and urease activities, (2) soil carbon/nitrogen ratio was positively related with β-1,4-glucosidase, cellobiosidase and peroxidase activities and (3) soil acid phosphatase activity was negatively related with total and available phosphorus and positively with nitrogen/phosphorus ratio, which suggest that soil carbon, nitrogen, phosphorus and their ratios were the most important factors influencing soil microorganism and enzyme activities. The change in tree species, short felling cycle, poor understory coverage and litter layer, field burning and tillage might be the factors that lead to decrease in soil nutrient, microbial biomass and enzyme activity in the soil, while fertilization might alleviate the effect on soil nutrients. Keeping proper understory coverage and litter layer, reducing soil disturbance and fertilizing properly would help improve soil quality during the replacement processes and follow-on management of the plantations and hence ensure sustainable development of Eucalyptus plantations.