Soil microorganisms serve as the pivotal biological agents driving the nitrogen biogeochemical cycle, with research into their community structure and functional genes holding significant scientific importance for revealing soil nitrogen metabolism processes. The karst forest ecosystem, a typical and fragile ecological barrier in southwestern China, is highly susceptible to external disturbances. Within karst forest soils, the conversion and cycling of nitrogen are particularly complex, involving the synergistic action of multiple microorganisms and the regulation of functional genes. To further elucidate the nitrogen metabolism pathways of soil microorganisms in karst forests, this study selected soil from the Maolan karst forest ecosystem in Guizhou Province as the research object. Typical soil samples were collected to analyze soil physicochemical properties. Macrogenomic technology was employed to identify nitrogen-metabolizing microorganisms and functional genes, thereby dissecting nitrogen metabolism pathways and further exploring the influence of environmental factors. The results indicated that the soil fertility in the Maolan karst forest was of high level, with an average total nitrogen content (4.32 g/kg) significantly higher than the national forest average (1.06 g/kg). The dominant nitrogen-metabolizing microorganisms identified were Bradyrhizobium, Mycobacterium, and Streptomyces, while the dominant nitrogen metabolism functional genes were nasA, norB, and narG. The nitrogen metabolism pathways were characterized by Dissimilatory nitrate reduction > Assimilatory nitrate reduction > Denitrification > Complete nitrification > Nitrification > Nitrogen fixation. Among them, the nitrogen-metabolizing microorganism and functional gene most affected by environmental factors were Methyloceanibacter and narB, respectively. The research findings further refine the theoretical framework of soil nitrogen cycling in karst forest habitats and provide vital data support and theoretical evidence for nitrogen regulation and microbial resource utilization in this regional ecosystem.