The change in land-use systems due to new management practices and confined agricultural space leads to a major shift in soil health. Soil microbial community composition, enzyme activities, and soil physiochemical properties are considered as important indicators of soil health. Past research has focused on soil health in response to major land-use systems such as forest, pasture, and cultivated land. However, the response of soil health indicators specifically, biological indicators to silvopasture systems remains understated. Therefore, the objective of our study was to assess the impact of silvopasture (SPS) and woodland (WS) land-use systems on the soil health indicators. The study site consists of 5-years-old southern-pine silvopasture developed from existing woodland and 13-years old southern-pine/hardwood mixed woodland plots. Soil samples were collected and analyzed for microbial analysis using the MiSeq platform next-generation sequencing, enzyme assay using a microplate fluorimetric method, and soil physiochemical properties. Significantly higher soil organic carbon (2.6 ± 0.12%), total nitrogen (0.09 ± 0.002%), Mehlich phosphorous (26.9 ± 3.09 mg kg−1), nitrate (4.8 ± 0.24 mg kg−1), and pH (6.5 ± 0.10) were observed in SPS. SPS had a significantly higher acidic phosphatase (169.5 ± 6.59), alkaline phosphatase (43.6 ± 3.39), phosphodiesterase (10.5 ± 0.71), and β-glucosidase (61.7 ± 3.34) activity, measured in nmol g−1 hr−1, as compared to WS. Proteobacteria, Actinobacteria, and Acidobacteria were the most dominant bacterial phyla while Ascomycota and Basidiomycota were the most dominant fungal phyla in both systems. The relative abundance of Proteobacteria (43.3%) was significantly higher in SPS while that of Acidobacteria (17.5%) was significantly higher in woodland (WS). Similarly, Basidiomycota were dominant (p < 0.0001) in WS while SPS was dominated by Ascomycota (p < 0.0001). Microbial diversity and the microbial community at the class and genus taxonomic levels were also impacted by the SPS. Copiotrophs and phytopathogens were higher in SPS while oligotrophs, decomposers, and ectomycorrhizae were higher in WS. In conclusion, our study found strong evidence that the nutrient input and vegetation composition in southern-pine SPS led to changes in soil physiochemical properties which further caused a shift in soil microbial community composition in these systems identified by principal component analysis.