Extensive afforestation with exotic species has caused broad concern due to its potential impacts on ecosystems. Eucalyptus is one of the main introduced tree species in China. Understanding the impacts of afforestation with Eucalyptus on the structure and function of soil microbial communities plays a key role elucidating the impacts of a single species on ecosystem services and implementing the scientific management in Eucalyptus plantations. Paired comparison (i.e. natural secondary forests and Eucalyptus plantations) test was used to evaluate how the soil microbial structure and function respond to land use change from natural secondary forests to exotic Eucalyptus plantations in Baisha County, Hainan Province, southern China. The soil microbial communities were evaluated by comparing the biomass, phospholipid fatty acid (PLFA) composition, and carbon metabolic function (BIOLOG profiles). Compared with those in natural secondary forests, the size of soil microbial communities in Eucalyptus plantations significantly decreased. The mean value of soil microbial biomass carbon in Eucalyptus plantations was 376.65 mg/kg, but it was 631.86 mg/kg in natural secondary forests. The mean values of soil microbial biomass nitrogen in Eucalyptus plantations and natural secondary forests were 61.91 mg/kg and 94.72 mg/kg, respectively. The soil microbial biomass in Eucalyptus plantations was significantly lower than that in natural secondary forests. Additionally, Eucalyptus plantations had significantly lower abundances of gram-positive bacterial, gram-negative bacterial, arbuscular mycorrhizal fungal, fungal, actinomycetic, and total phospholipid fatty acids than the natural secondary forests. Forest conversion from natural secondary forests to Eucalyptus plantations resulted in an increase in soil microbial physiological stress, and a decrease in soil microbial function. The indicators denoting stress related to soil nutrient levels, the ratios of monounsaturated to saturated fatty acids, gram-positive to gram-negative bacteria, iso- to anteiso-branched PLFA, and cy19:0 to 18:1ω7c in Eucalyptus plantations significantly increased after the forest conversion. However, the soil microbial carbon metabolic activity, richness, and diversity significantly decreased. The significant changes in soil microbial community structure and function after the forest conversion from natural secondary forests to Eucalyptus plantations were mainly due to the decreases of plant species richness and coverage, and soil carbon, nitrogen and water resource availability. Canonical correspondence analysis (CCA) indicated that the significant differences in the PLFA composition and carbon utilization profiles of soil microbial communities were mainly due to the species richness and coverage of the tree layer, species richness of the shrub layer, soil water content, soil organic carbon, soil pH, and soil alkaline hydrolyzable-nitrogen. In conclusion, forest conversion from natural secondary forests to exotic Eucalyptus plantations resulted in the deterioration of soil microbial communities. Improved management practices, such as reducing soil or understory disturbance during logging, and the subsequent establishment of the next rotation plantation, should be considered to help improve the structure and function of soil microbial communities and increase soil resource availability during plantation management.