Abstarct: Abstract Glycyrrhiza glabra L. is one of the valuable medicinal plants, and the glycyrrhizin present in its root has wide applications in the food and pharmaceutical industries. This research was conducted with the aim of investigating the effect of biotic stimulants including arbuscular mycorrhizal fungus (Rhizophagus irregularis) and two plant growth-promoting bacterial species, Pantoea agglomerans (P5) and Pseudomonas putida (P13), along with drought stress at three levels of field capacity 80 (FC80), 60 (FC60), and 40 (FC40) percent, on growth traits, physiological characteristics, glycyrrhizin content, and the exxpression of key genes in its biosynthesis pathway. The experiment was conducted as a factorial arrangement in a randomized complete block design with 3 replications. The results showed that drought stress caused a significant increase in glycyrrhizin content; such that in FC60 and FC40 treatments, 42 and 73 percent increase compared to the control (FC80) were observed, respectively. Also, biotic treatments, especially the combination of mycorrhizal fungus with P5 bacterial strain, led to an increase in dry weight of plant up to 25% and an increase in number of leaves up to 30%. Simultaneous application of fungus and bacteria caused a 23% increase in root length and improvement of 28 and 31% in phosphorus uptake in leaf and root, a 45% increase in soluble sugar, and a 35% decrease in electrolyte leakage. Under mild stress, the treatment containing fungus and Pseudomonas putida P13 strain increased leaf flavonoid content and root diameter by 62.2 and 46.7times compared to the control, respectively. In gene exxpression analysis, the mycorrhizal fungus treatment along with Pseudomonas putida bacteria under stress conditions caused a significant increase in the exxpression of HMGR and CYP88D6 genes. While the exxpression of β-AS and SQS genes was highest in the treatment containing fungus and Pantoea agglomerans P5 strain under moderate drought stress conditions. Also, the highest exxpression of the CYP72A154 gene was observed in the fungus-only treatment under non-stress conditions. In general, the results of this study showed that the combined application of biological agents and mild drought stress can, in addition to improving plant growth, enhance the glycyrrhizin biosynthesis pathway and increase the production of this valuable mextabolite.