Microbial communities in subsurface soil are specialized for their environment, which is distinct from that of the surface communities. However, little is known about the microbial communities (bacteria and fungi) that exist in the deeper soil horizons. Vertical changes in microbial alpha-diversity (Chao1 and Shannon indices) and community composition were investigated at four soil depths (0–10, 10–20, 20–40, and 40–60 cm) in a natural secondary forest of Betula albosinensis by high-throughput sequencing of the 16S and internal transcribed spacer rDNA regions. The numbers of operational taxonomic units (OTUs), and the Chao1 and Shannon indices decreased in the deeper soil layers. Each soil layer contained both mutual and specific OTUs. In the 40–60 cm soil layer, 175 and 235 specific bacterial and fungal OTUs were identified, respectively. Acidobacteria was the most dominant bacterial group in all four soil layers, but reached its maximum at 40–60 cm (62.88%). In particular, the 40–60 cm soil layer typically showed the highest abundance of the fungal genus Inocybe (47.46%). The Chao1 and Shannon indices were significantly correlated with the soil organic carbon content. Redundancy analysis indicated that the bacterial communities were closely correlated with soil organic carbon content (P = 0.001). Collectively, these results indicate that soil nutrients alter the microbial diversity and relative abundance and affect the microbial composition.