Biological soil crusts (BSCs), a vital component of ecosystems, are pivotal in carbon sequestration, nutrient enrichment, and microbial diversity conservation. However, their impact on soil microbiomes in alpine regions remains largely unexplored. Therefore, this study aimed to determine the influence of BSCs on alpine grassland soil microbiomes, by collecting 24 pairs of soils covered by biological and physical crusts along a transect on the Qinghai-Tibetan Plateau. We found that BSCs significantly increased the contents of soil moisture, organic carbon, total nitrogen, and many available nutrients. They also substantially altered the soil microbiomes. Specifically, BSCs significantly increased the relative abundance of Cyanobacteria, Verrucomicrobiota, and Ascomycota, while decreasing the proportions of Gemmatimonadota, Firmicutes, Nitrospirae, Mortierellomycota, and Glomeromycota. By contrast, microbial abundance and α-diversity demonstrated low sensitivity to BSCs across most study sites. Under the BSCs, the assembly of prokaryotic communities was more affected by homogeneous selection and drift, but less affected by dispersal limitation. Conversely, soil fungal community assembly mechanisms showed an inverse trend. Overall, this study provides a comprehensive understanding of the effects of BSCs on soil properties and microbial communities, offering vital insights into the ecological roles of BSCs.