In desert ecosystems, the desertification process is characterized by increasing attenuation of plant productivity and deterioration of soil habitats, leading to enhanced environmental stress gradients for soil microbiomes. Despite the significance of microbial communities for multifunctionality in terrestrial ecosystems, the feedback dynamics of microbiomes and their contributions to maintaining subsurface soil multifunctionality as desertification progresses have yet to be evaluated. Here, we used three sites with different desertification stages and investigated the variation trends of microbiomes in soil profiles (0-100 cm) and their contributions to regulating multifunctionality. We first confirmed that multifunctionality did not exhibit a significant difference between superficial soils (0-20 cm) and deep soils (20-100 cm) and slightly decreased as soil depth increased throughout the entire profile. Desertification progression drove distinct variation trends of microbiomes in vertical soil profiles. Soil bacterial communities received on average more positive and progressive feedback from desertification development than fungal and archaeal communities, characterized by significant variation in bacterial alpha- and beta-diversity and slight variation in fungal and archaeal alpha- and beta-diversity. The most abundant phyla in the microbiomes did not vary between the superficial and deep soils at any desertification stage. Significant declines in microbial clades within Acidobacteria are an important feature as desertification proceeds. Particular microbial taxa rather than total microbial diversity best predict and explain the vertical profile variation in soil multifunctionality in desert ecosystems. Our results highlight the significance of microbial community composition in subsurface soils for regulating multifunctionality in desert ecosystems.