China has experienced a widespread increase in N deposition due to intensive anthropogenic activities, particularly in the subtropical regions. However, the effects of long-term N deposition on soil bacterial and fungal abundance, diversity, and community composition remain largely unclear. We assessed the effects of N deposition on soil microbial communities in summer and winter, using quantitative polymerase chain reaction and Illumina Miseq sequencing of bacterial 16S rRNA and fungal ITS genes from subtropical natural forest soils. The abundance of both soil bacteria and fungi exhibited a decreasing pattern with increasing N deposition rates. Nitrogen deposition increased bacterial diversity in both summer and winter, whereas fungal diversity was significantly decreased in summer, but greatly increased under the highest level of N deposition (150 kg N ha⁻¹ yr⁻¹) in winter. Nitrogen deposition significantly increased the relative abundance of bacterial phyla Actinobacteria, Chloroflexi, and WPS-2, but decreased that of Acidobacteria and Verrucomicrobia. In addition, N deposition significantly decreased the relative abundance of Ascomycetes, but did not exert any significant effect on Basidiomycetes. The bacterial and fungal community compositions were greatly influenced by N deposition, with soil N availability and soil pH identified as the two most influential soil properties. This study demonstrates that the fungal community was more sensitive than the bacterial community to N deposition, and further emphasizes the importance of simultaneously evaluating soil bacterial and fungal communities in response to global environmental changes.