Climatic change causes obvious seasonal meteorological drought in southern China, yet there is a lack of comprehensive in situ studies on the effects of drought in Eucalyptus plantations. Here, a 50% throughfall reduction (TR) experiment was conducted to investigate the seasonal variations of soil bacterial and fungal communities and functions in a subtropical Eucalyptus plantation and their responses to TR treatment. Soil samples were collected from control (CK) and TR plots in the dry and rainy seasons and were subjected to high-throughput sequencing analysis. Results showed that TR treatment significantly reduced soil water content (SWC) in the rainy season. In CK and TR treatments, fungal alpha-diversity decreased in the rainy season while bacterial alpha-diversity did not change significantly between dry and rainy seasons. Moreover, bacterial networks were more affected by seasonal variations compared with fungal networks. Redundancy analysis showed that alkali hydrolyzed nitrogen and SWC contributed the most to the bacterial and fungal communities, respectively. Functional prediction indicated that the expression of soil bacterial metabolic functions and symbiotic fungi decreased in the rainy season. In conclusion, seasonal variations have a stronger effect on soil microbial community composition, diversity, and function compared with TR treatment. These findings could be used to develop management practices for subtropical Eucalyptus plantations and help maintain soil microbial diversity to sustain long-term ecosystem function and services in response to future changes in precipitation patterns.