The impact of forest microhabitats on physiochemical properties of the soil and that of microbial communities on tropical soils remain poorly understood. To elucidate the effect of tropical forest stand on leaf litter and soil microbial communities, we studied enzyme activities, microbial biomass, and diversity in three distinct microhabitats in terms of plant richness, diameter at breast height (DBH), and physiochemical properties of soil and litter, each associated with a different Vanilla sp. In the soil, positive correlations were found between electrical conductivity (EC) and total organic carbon (TOC) with phosphatase activity, and between nitrogen (N) and water-soluble carbon (WSC) content with urease activity (UA). In the litter, the water content was positively correlated with bacterial and fungal biomass, and N and WSC contents were positively correlated with fungal biomass. Positive correlations were found between plant richness and UA in the soil, plant richness and fungal biomass in the soil and litter, and DBH and fungal biomass in the litter. Amplicon sequencing revealed differences between microhabitats in the relative abundance of some fungal and bacterial taxa and in the bacterial community composition of both litter and soil. Bacterial richness and diversity were different between microhabitats, and, in litter samples, they were negatively correlated with DBH and plant richness, respectively. By contrast, none of the soil and litter physiochemical properties were significantly correlated with microbial diversity. Our results show that significant shifts in enzyme activity, microbial biomass, and diversity in the microhabitats were driven by key abiotic and biotic factors depending on the soil or litter sample type.