Soil is a complex and dynamic biological system. Agroforestry systems are considered to be an alternative land use option to help and prevent soil degradation, improve soil fertility, microbial diversity, and organic matter status. An increasing interest has emerged with respect to the importance of microbial diversity in soil habitats. The present study deals with the status of microbial diversity in agroforestry systems in Tamil Nadu. Eight soil samples were collected from different fields in agroforestry systems in Cuddalore, Villupuram, Tiruvanamalai, and Erode districts, Tamil Nadu. The number of microorganisms and physico-chemical parameters of soils were quantified. Among different microbial population, the bacterial population was recorded maximum (64%), followed by actinomycetes (23%) and fungi (13%) in different samples screened. It is interesting to note that the microbial population was positively correlated with the physico-chemical properties of different soil samples screened. Total bacterial count had positive correlation with soil organic carbon (C), moisture content, pH, nitrogen (N), and micronutrients such as Iron (Fe), copper (Cu), and zinc (Zn). Similarly, the total actinomycete count also showed positive correlations with bulk density, moisture content, pH, C, N, phosphorus (P), potassium (K), calcium (Ca), copper (Cu), magnesium (Mg), manganese (Mn), and zinc (Zn). It was also noticed that the soil organic matter, vegetation, and soil nutrients altered the microbial community under agroforestry systems.