Biocrusts occupy the surface of soils, house key microbes and non-vascular plants, and further provide other vital ecological functions in oligotrophic drylands. However, little is known about the impact of trampling on fragile biocrusts in temperate deserts, particularly changes to soil microbial communities. To examine this, fresh soils from three trampled intensities to biocrusts were sampled in vegetation areas of the Tennger Desert. Soil microbial biomass carbon and nitrogen, and microbial communities were studied using chloroform fumigation and Illumina sequencing, respectively. The results collected 2050 OTUs and 393 species of bacterial communities and 1124 OTUs and 135 species of fungal communities. Severe trampling of biocrusts resulted in a reduction in soil microbial biomass carbon and nitrogen, and soil microbial abundance and diversity; changed the relative abundance of microbial taxa; altered the soil microbial community structures of cyanobacteria-dominated crusts, and further affected microbial community functions. Reduced soil moisture and nutrients and enhanced pH were the factors which caused alteration in soil microbial communities after trampled biocrusts. In addition, there was a negative correlation between trampling intensity with soil nutrient content, soil microbial biomass carbon and nitrogen, and microbial abundance. After similar trampling intensity, later-succession moss-dominated crusts were significantly higher in soil nutrients, soil microbial biomass carbon and nitrogen, soil fungal abundance and diversity, and more distinctive microbial community structures compared to early-succession cyanobacteria-dominated crusts. This suggests that there is a positive correlation between biocrust tolerance to trampling and the successional stages of biocrusts. Therefore, severe trampling of biocrusts could modify biocrust fragile structures and functions, which in turn, altered soil microbial community compositions and structures, and discouraged their function, leading to a degradation process of surface soils in temperate desert ecosystems.