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The aim of this study was to evaluate the relationships among changes in the soil microstructure, soil physicochemical traits and microbial communities during desertification. Soil samples were collected from grasslands at different stages of desertification, from an area of the Tibetan Plateau. Soil aggregate fractionation and soil physicochemical properties were analysed using standard methods, and the abundance and diversity of bacteria associated with different aggregate fractions were estimated by quantitative PCR and Illumina Miseq amplicon sequencing. Sequential changes in response to desertification stages were detected in the analysed features. For instance, soil nutrients, microbial biomass C and N, water content and pH initially changed during the light desertification stage; then, the soil aggregation decreased during the intermediate stages; and finally, bacterial abundance and diversity decreased during heavy and severe desertification stages. During grassland desertification, vegetation coverage positively influenced soil aggregation by affecting soil properties (e.g., pH and SOC), whereas bacterial communities in FMi (free microaggregates) and FSC (free silt and clay) were negatively related to soil aggregation. This study reports novel information concerning the desertification process and the sequential modification of soil traits and interactions among soil physical properties (aggregate composition), soil nutrients and the soil microbial community in the grasslands of the Tibetan Plateau. Highlights: Bacterial community was more sensitive in macroaggregates than in microaggregates.Composition of small macroaggregates and associated bacteria changed during desertification.Bacterial community in microaggregates, silt and clay negatively affected aggregation.Vegetation coverage affected aggregation by changing soil traits and bacterial community. [ABSTRACT FROM AUTHOR]