<p>To investigate the effects of different land‑use practices on the soil ecosystem in the Huaihe River source region, this study selected forestland, farmland, and nursery land in Tongbai County, Henan Province, as research objects. A combination of high‑throughput sequencing and soil physicochemical analysis was employed to systematically compare the soil microbial community structure, diversity, and their relationships with key environmental factors among the three land‑use types. The results showed significant differences in soil physicochemical properties: farmland had the highest contents of available phosphorus (AP) and available potassium (AK), forestland had significantly higher AP content than nursery land (<i>P &lt;</i> 0.05), while nursery land exhibited significantly higher soil water content (SWC) and bulk density (SBD) than the other two land‑use types (<i>P &lt;</i> 0.05). Microbial diversity analysis revealed that the bacterial Simpson index and the fungal Shannon and Simpson indices were relatively higher in farmland soil (<i>P &lt;</i> 0.05). Redundancy analysis indicated that soil organic matter (SOM) was the key environmental factor driving changes in both bacterial and fungal community structures. In terms of community composition, Acidobacteriota and Ascomycota were the dominant phyla for bacteria and fungi, respectively. Functional prediction analysis further revealed that the biosynthesis pathway was the most prominent metabolic function in both microbial communities. In summary, land‑use practices significantly altered the soil physicochemical properties and microbial communities in the Huaihe River source region. Moderate agricultural use maintained relatively high microbial diversity, and its community structure was mainly regulated by soil organic matter. This study provides a scientific basis for understanding soil ecological processes and the sustainable management of land resources in this region.</p>