Soil water content and salinity critically regulate soil microbial composition, plant community structure, and ecosystem multifunctionality (EMF) in semi-arid grasslands. However, the mechanisms through which drought (D), saline-alkaline (SA), and their combined (DSA) stress influence these ecological components remain poorly understood. This study investigated these mechanisms along natural gradients in a semi-arid grassland of China by analyzing soil physical-chemical properties, microbial communities, and vegetation characteristics. The results showed that as the environmental stress shifted from the D group to the DSA group and then to the SA group, soil electrical conductivity significantly increased, while urease and phosphatase activities significantly decreased. Soil organic carbon, total nitrogen, total phosphorus, and microbial biomass carbon and nitrogen were lower in the D and SA groups than in the DSA group. Meanwhile, plant biomass showed an increasing trend along the treatment gradient, primarily driven by dominant species, while plant diversity did not exhibit significant differences. Further analysis identified the soil water content and salinity as the key determinants of soil microbial diversity and community complexity. Soil enzyme activities exhibited contrasting relationships with microbial composition, correlating positively with the richness of bacterial amplicon sequence variants (ASVs) but negatively with the richness of fungal ASVs. Notably, microbial biomass, which varied significantly across different groups, emerged as a key predictor of changes in EMF, with its critical role confirmed through structural equation modeling. These findings collectively elucidate the responses of ecological communities to synergistic soil hydro-saline stress in semi-arid ecosystems, while highlighting the critical role of microbial biomass in maintaining EMF.