Soil microorganisms play key roles in ecosystems and respond quickly to environmental changes. Liming and/or understory removal are important forest management practices and have been widely applied to planted forests in humid subtropical and tropical regions of the world. However, few studies have explored the impacts of lime application, understory removal, and their interactive effects on soil microbial communities. We conducted a lime application experiment combined with understory removal in a subtropical Eucalyptus L’Hér. plantation. Responses of soil microbial communities (indicated by phospholipid fatty acids, PLFAs), soil physico-chemical properties, and litter decomposition rate to lime and/or understory removal were measured. Lime application significantly decreased both fungal and bacterial PLFAs, causing declines in total PLFAs. Understory removal reduced the fungal PLFAs but had no effect on the bacterial PLFAs, leading to decreases in the total PLFAs and in the ratio of fungal to bacterial PLFAs. No interaction between lime application and understory removal on soil microbial community compositions was observed. Changes in soil microbial communities caused by lime application were mainly attributed to increases in soil pH and NO3–-N contents, while changes caused by understory removal were mainly due to the indirect effects on soil microclimate and the decreased soil dissolved carbon contents. Furthermore, both lime application and understory removal significantly reduced the litter decomposition rates, which indicates the lime application and understory removal may impact the microbe-mediated soil ecological process. Our results suggest that lime application may not be suitable for the management of subtropical Eucalyptus plantations. Likewise, understory vegetation helps to maintain soil microbial communities and litter decomposition rate; it should not be removed from Eucalyptus plantations.