Soil microbes are involved in many important ecosystem processes including nutrient acquisition, biogeochemical cycling and soil aggregation. Soil microbial diversity affects the soil belowground dynamics and fate of carbon and nutrients. Soil microbes are important for agricultural and plant production systems, hence understanding the effects of agroforestry systems on the soil microbes, is necessary in order to improve on soil health and fertility. The objective of the study was to determine the soil microbial biomass, microbial populations and microbial diversity in maize-banana based agroforestry system. The study was conducted at Kenya Agricultural and Livestock Research Organization farm in Kisii County. The experiment was laid out in a randomized complete block design with maize and banana intercropped with agroforestry trees. The treatments were; Maize, banana (MMBB), Maize-banana, Calliandra (MBCC), Maize (MM), banana (BB), Maize-banana, Leucaena (MBLL), Maize-banana, Sesbania (MBSS) and Maize, fertilizer (MMF). Soil samples were   collected from the agroforestry fields using a soil auger. Soil microbial biomass was measured using the chloroform fumigation extraction. Fungi and bacteria were enumerated by serial dilution plate method.Shannon diversity index (H’) and Simpson diversity index (1 - D) were used for the calculation of species diversity. SAS (version 9) statistical software was used for analysis. The treatments with agroforestry tree species had significantly higher soil microbial biomass (MBSS-86.33, MBCC-52.66 and MBLL- 47.0MgC/Kg) populations of bacteria (MBSS-197, MBCC-128.0 and MBLL-111.25x108cfu g-1soil) and fungi (MBSS-50.83, MBCC-29.167 and MBLL-14.0x105cfu g-1 soil) and diversity of bacteria (MBSS- (H' =1.61, D = 1), MBCC- (H' =1.04, D = 0.83), MBLL (H' = 0.52, D = 0.5) and fungi MBSS (H' =1.39, D = 1) MBCC (H' =1.04, D= 0.83), MBLL (H' =1.56, D = 0.93). MBSS increased microbial biomass, microbial populations and microbial diversity significantly an indication of improved soil health and hence recommended for adoption by farmers.