Abstract: Drought, one of the major abiotic stresses affecting plant growth and development, severely restricts crop productivity. This study aimed to screen plant growth-promoting rhizobacteria (PGPR) with excellent growth-promoting and drought-resistant traits from the rhizosphere soil of Onobrychis viciifolia (sainfoin) in different drought habitats, and to evaluate their regulatory effects on the growth, development, and physiological responses of Medicago sativa seedlings under drought stress. A total of 903 culturable bacterial strains belonging to 82 species were isolated via multi-medium cultivature and identified via 16S rRNA gene sequencing. These strains were predominantly affiliated to the phyla Proteobacteria, Actinobacteria, and Bacteroidetes, with the genera Serratia and Pseudomonas being dominant. Analysis of plant growth-promoting traits revealed that the 82 rhizobacterial strains exhibited varying degrees of plant growth-promoting properties. Specifically, strains OvR165 (Enterobacter sp.), OvR548 (Acinetobacter sp.) and OvR687 (Pseudomonas sp.) possessed the capabilities of phosphate solubilization, nitrogen fixation, siderophore production, indole-3-acetic acid (IAA) secretion and extracellular polysaccharide (EPS) synthesis. Among them, OvR548 and OvR687 also exhibited strong biofilm formation abilities. Pot experiments demonstrated that inoculation with these three strains significantly alleviated the inhibitory effects of drought on Medicago sativa seedlings. The inoculation of the strains increased the plant height, root length, leaf area, biomass accumulation, chlorophyll content, and relative water content, while reducing the cell membrane damage and reactive oxygen species (ROS) accumulation, thus effectively mitigating drought stress-induced damage to plants. Additionally, these strains showed strong potential for improving plant tolerance to drought, further enhancing the ecological adaptability of crops under water scarcity conditions. In conclusion, the rhizosphere of O. viciifolia in different arid regions of Qinghai Province harbors abundant and functionally diverse resources of plant growth-promoting and drought-tolerant bacteria, which have great ecological value and application prospects. This study provides excellent strain resources and theoretical support for sustainable grassland management and microbial agent development in arid regions, helping to improve the productivity and resilience of crops in drought-prone areas.