It is well known that biodiversity and ecosystem multifunctionality (EMF) guarantee the well-being of human society. Most studies have focused on the relationship between biodiversity and ecosystem function, and less is known about the individual and combined effects of above- and below-ground biodiversity on ecosystem multifunctionality under grazing disturbance. The aim of our study was to investigate the relationship between plant and soil microbial (bacterial and fungal) diversity and ecosystem multifunctionality under grazing disturbance by using multiple methods to assess ecosystem multifunctionality. We conducted experiments in desert grasslands on the northern slopes of the Tian Shan Mountains and compared the relationship between ecosystem multifunctionality and biodiversity assessed by different methods under light grazing and heavy grazing. Our results showed that at the heavy grazing level, ecosystem multifunctionality calculated by the mean method and plant diversity, soil fungal diversity, soil bacterial diversity and soil fertility calculated by the single function method showed a significant decrease (p < 0.05), but grass productivity was significantly increased (p < 0.05). Among them, ecosystem multifunctionality, soil carbon storage function and soil fertility all showed significant positive correlations with plant diversity and soil microbial diversity (p < 0.05). We calculated that ecosystem multifunctionality also essentially showed positive correlation with plant diversity and soil microbial diversity using the multi-threshold method, and the effect curve was approximately a single-peaked curve, first increasing and then decreasing. Finally, we used plant diversity, soil fungal diversity and soil bacterial diversity under grazing disturbance as biotic factors and soil pH as an abiotic factor to construct structural equation models, and we found that grazing can have direct effects on ecosystem multifunctionality and indirect effects on ecosystem multifunctionality through above- and below-ground biodiversity. Our study emphasizes the importance of the combination of above- and below-ground biodiversity in maintaining the multifunctionality of desert grassland ecosystems on the northern slopes of the Tian Shan Mountains. A moderate reduction in grazing intensity can better conserve biodiversity and improve ecosystem multifunctionality, and it is a feasible strategy to maintain sustainable management of desert grasslands.