Understory removal and/or nitrogen fertilization are important forest management practices in forest plantations. Relative to overstory vegetation, understory vegetation is overlooked in forest ecosystems; and the effects of nitrogen fertilization on soil microbial communities are poorly understood. The objective of this study was to improve our knowledge of how understory removal and/or fertilization influence soil microbial communities. We conducted an experiment in which understory was removed or retained in plots with or without nitrogen fertilization in eucalyptus plantations; and we measured their effects on soil temperature, soil water content, and soil microbial communities (as indicated by phospholipid fatty acids). Understory removal increased soil temperature, decreased soil water content, decreased fungal biomass and decreased fungal to bacterial biomass (F:B) values. Additionally, fungal biomass correlated negatively with soil temperature. Nitrogen fertilization did not significantly affect soil temperature, soil water content, or soil microbial community. These findings suggest that the presence of understory is favorable for sustaining soil microclimates and acts as an important driver of soil microbial communities in eucalyptus plantations. Moreover, understory vegetations are important components and should not be removed from eucalyptus plantations.