Tropical rainforest soils, or latosols, are distinguished by their low pH and low fertility. In orchards, co-cultivating
grass has become popular as a way to improve soil quality and boost fruit production. Nevertheless, insufficient
information is currently available about the response of soil microbial communities in tropical rainforest orchards to
grass co-cultivation. Therefore, the present research investigates the effect of grass cultivation on the soil properties
and microbial diversity of guava (Psidium guajava L. cv Pearl) latosol orchards. Two varieties of the tropical legume
grass Stylosanthes guianensis, i.e., Reyan No. 2 and Ubon, were studied, besides the control (CK), which is without
any grass, and the natural grasses treatment (N). The study contained four treatments, i.e., S. guianensis cv. Reyan
No. 2, S. guianensis cv. Ubon, CK, and N. Soil samples from the top layer (0–20 cm) and subsoil layer (20–40 cm)
were collected to follow the changes in soil microbial biodiversity based on 16 S rDNA analysis. A total of 17,231
kinds of OTUs (Operational Taxonomic Units) were obtained, including 17,165 kinds of bacteria and 66 kinds of
Archaea. S. guianensis cv. The Ubon variety, natural grasses, and CK treatments significantly increased the soil
microbial richness and evenness in the topsoil layer compared to Reyan No. 2 variety. The β-diversity of soil
microbial community was significantly reduced in the natural grasses and Ubon variety treatments at the topsoil
layer compared to CK treatment. In the subsoil layer, natural grasses, Reyan No. 2, and Ubon treatments significantly
increased the soil microbial community based on β-diversity. The presence of natural grasses caused 49% and 42%
increases in the SOC in the top and subsoil layers, respectively, as well as remarkable increases in the available and
total soil nitrogen. The grass intercropping enhanced the levels of soil carbon and nitrogen and altered the nature of the soil’s microbial community. The diversity of soil microorganisms in the subsoil layer is significantly altered by
the shallow root systems of tropical legume and natural grasses, which have most of their roots concentrated in
the top soil layer. Overall, growing grass in tropical orchards benefits the latosolic soil microorganisms, which has
enhanced the theoretical underpinnings for using grass to improve the soil quality in latosols orchards.