Background and aims <p>The spontaneous secondary succession of degraded grasslands represents a pivotal mechanism for the restoration of ecosystem functions, yet its impact on the soil microbial community structure within zokor-disturbed grasslands remains inadequately characterized.</p> Methods <p>This study elucidates soil microbial dynamics across successive stages of zokor-disturbed grassland on the Qinghai-Tibetan Plateau using high-throughput sequencing technology and a space-for-time substitution framework.</p> Results <p>Our investigation reveals bacterial alpha-diversity augments while fungal alpha-diversity declines as succession progresses. Notably, bacterial communities exhibited heightened sensitivity to the recovery process relative to fungal counterparts, evidenced by effect sizes of 0.134 in relation to soil environments. Soil moisture emerged as the principal determinant of microbial community composition. Through ecological network analysis, we identified a preponderance of positive associations among microbial taxa, suggestive of niche-sharing dynamics. Additionally, vegetative carbon content and coverage were identified as the strongest phylogenetic signals influencing bacterial and fungal taxa, respectively. Phylogenetic conservatism was found to govern the soil bacteria, in contrast to the more dispersed distribution patterns observed in soil fungi.</p> Conclusions <p>These findings underscore the distinct responses of bacterial and fungal communities to environmental changes during grassland succession and highlight the importance of soil moisture and ecological interactions in shaping microbial dynamics.</p>