Karst mountain areas are typically known as ecologically fragile areas in China. There are a variety of microhabitats that are distributed over the area, including stone grooves, stone gullies, top-soils, stone caverns, and stone crevices. Soil microorganisms are the most active components of terrestrial ecosystems, and they promote energy flow and material circulation through the ecosystem. Studies that examine the impact of different types of microhabitats on the composition of soil microbial communities have great significance for the better understanding of the microbial characteristics of karst forest in mountain area and the karst ecological protection. With the Maolan Karst Forest microhabitats as the research subjects, this study comparatively analyzed soil physical and chemical properties and microbial composition, and it quantitatively clustered the microhabitats. The results showed that 48 phospholipid fatty acids (PLFAs) were detected in the soils of six microhabitat types, and among these, there were 18 unique PLFAs that characterized microbes. The contents of total microbes, bacteria, fungi and actinomycetes, G⁺, and G^- in all microhabitat types were in the following order:stone groove > stone gully > top-soil > stone cavern > stone crevice > sub-soil. Moreover, the microbial contents in all habitats were in the following order:bacteria > actinomycetes > fungi. The values of G⁺/G^- in all habitats were as follows:stone groove > stone gully > top-soil > stone crevice > stone cavern > sub-soil. The ratios of fungi/bacteria ranged from 0.14 to 0.16, and the ratios were ordered as follows:top-soil > stone crevice > stone gully > stone groove > stone cavern > sub-soil. Correlation and Multivariate Regression Tree (MRT) analyses showed that soil organic matter from a different microhabitat was the primary factor affecting soil microbial community composition. The results of MRT clustering showed that the karst forest microhabitat can be divided into four types:stone groove-stone gully type, stone cavern-stone crevice type, top-soil type, and sub-soil type. The results confirmed that microhabitat types had significant impacts on soil microbe distribution in the karst forest.