Abstract: Background and Objective: Soil biological activity has been influenced by several biotic and abiotic factors prevailing in the region. For instance, variation in species composition, management practices and climatic conditions has had their reflections on the microbial population vis-à-vis biomass in the agroforestry systems. The present study was designed to understand the seasonal dynamics of microbial population and microbial biomass under the agroforestry systems of sub-humid Gujarat, India. Materials and Methods: Seasonally collected soil samples (0-15 and 15-30 cm) have been used for analyzing their physicochemical properties. Moist samples were used for soil biological studies. Bacterial and fungal counts were made following serial dilution methods. Microbial biomass (C, N and P) were estimated following the chloroform-fumigation extraction method. Results: Agri-silvicultural and home garden systems registered greater bacterial (27.20×104 g–1) and fungal (75.86×102 g–1) counts in the topsoil (0-15 cm). More or less similar trend was also observed in microbial biomass carbon. However, microbial biomass nitrogen (50.71 μg μ–1) and phosphorus (5.83 μg μ–1) were highest in the home gardens. Seasonally, microbial counts and biomass (C, N and P) in the soil were maximum during spring and minimum during the rainy season. Conclusion: Soil microbial population (bacteria and fungi) and microbial biomass C, N and P were significantly (p<0.05) influenced by different typologies of agroforestry practices in the sub-humid tropics. Overall, our study concludes that the home garden system performed better in influencing the biological health in tree-based farming systems.