The landscape of the drylands is characterized by patches of shrub plants and various successional stages of biocrusts, creating microhabitats that influence soil microbial communities. While previous studies have primarily focused on soil prokaryotes and a limited subset of eukaryotes-mainly fungi-a critical knowledge gap remains: the intrinsic effects of different microhabitat types on the diversity, community composition, and assembly processes of total soil eukaryotes are still poorly understood. To address this gap, we investigated how soil depth, biocrust succession, and shrub cover influence the structure and assembly of total soil eukaryotic communities in the Gurbantunggut Desert, China. 18S rRNA gene-based high-throughput sequencing was used to analyze variations in soil eukaryotic communities among different microhabitats. All three microhabitat factors significantly influenced soil eukaryotic communities. Specifically, alpha diversity was higher in the subsoil layer, early-stage biocrusts, and in areas without shrub cover compared to the top crust layer, late-stage biocrusts, and areas with shrub cover, respectively. Archaeplastida and Opisthokonta emerged as the dominant eukaryotic groups. Network complexity was greater in the top crust, late biocrust stages, and under shrub-free conditions than in the subsoil, early-stage biocrusts, and areas with shrub cover. Community assembly processes shifted from homogeneous to heterogeneous selection from the subsoil to the surface crust and from early to late biocrust stages, whereas a shift from homogeneous selection to ecological drift occurred from shrub-covered to shrub-free areas. Soil pH, total organic carbon, and total phosphorus were identified as key drivers of eukaryotic community variation. Overall, our findings highlight that microhabitat heterogeneity, through its influence on soil nutrient status, plays a critical role in structuring soil eukaryotic communities in desert ecosystems. This study advances our understanding of how multiple microhabitat factors jointly shape microbial community dynamics in drylands.