This study aimed to investigate the role of plantation forests and natural secondary forests in controlling soil physicochemical properties and microbial biomass in urban forest ecosystems. (1) Background: Urban forests provide numerous benefits to urban ecosystems, but the interaction between forest stands and soil properties in controlling soil microbial biomass carbon (MBC) and nitrogen (MBN) remains poorly understood. The objective of this study was to examine how different forest types (plantation forests and natural secondary forests) influence soil physicochemical properties and microbial biomass in urban forest ecosystems. (2) Methods: We conducted a study in Shushan Urban Forest Park, Hefei, China, utilizing redundancy analysis and linear regression analyses to identify key environmental factors affecting the microbial distribution and significant correlations between soil properties and microbial biomass. (3) Results: Plantation forests generally had lower pH, water content, and organic carbon and nutrient content than natural forests. Natural forests exhibited higher microbial biomass and nutrient cycling capacity. Soil depth and forest type have significant effects on soil properties and microbial biomass in both growing and dormant seasons, with practical implications for forest management and soil conservation in similar ecosystems. Soil water content (SWC), pH, total nitrogen (TN), total phosphorus (TP), and soil organic carbon (SOC) were identified as key factors affecting microbial carbon and nitrogen distribution during both growing and dormant seasons. Our study provides important insights into the role of forest stands and soil physicochemical properties in controlling soil microbial biomass in urban forest ecosystems. Effective forest management strategies should be developed to promote sustainable and resilient forest ecosystems. Future research should investigate the underlying mechanisms driving these relationships and focus on promoting sustainable and resilient urban forest ecosystems.