The removal of litterfall in the Eucalyptus plantations in south China affected the plant biodiversity in these ecosystems was found based on the field observation and lab analysis. The protection times of species diversity of three Eucalyptus communities were different (Community I with no protection, Community II with 7-year-protection, Community III with 35-year-protection). The total numbers of species in these communities (from Community I to II to III) are 1, 6, and 17, respectively. The results showed that the protection of litterfall from being taken out of the ecosystem is important and can increase plant species diversity. This study combined biomass data, the chemical and physical properties of the soil, and the diversity of microbes in the communities. It is concluded that the mechanism of the effects of litter removal on biodiversity includes three factors: removing the suitable habitat of microbe and animal, decreasing the soil nutrient, and changing the special habitat for the germination and growth of invading plants. These results should have important implications for managing these Eucalyptus forest ecosystems in South China.