Drought is one of the most important factors worldwide, which limits the crop production, especially in semi-arid areas. The use of beneficial microorganisms such as arbuscular mycorrhizal fungi (AMF) may represent an eco-friendly and biological technique to increase crops yields and ensure food security. The purpose of this paper was to evaluate the effect of AMF inoculation on a promised variety of hulless barley (Hordeum vulgare ssp. nudum L.) under three levels of water stress (well-watered, moderate drought and severe drought). Hulless barley plants were inoculated, or not, with autochthonous inoculum (AI) containing five native AMF species (Pacispora franciscana, Funneliformis mosseae, Funneliformis geosporum, Rhizophagus irregularis and Glomus tenebrosum), or commercial inoculum (CI) containing Glomus sp. strains. Under water stress, AMF inoculation especially, with autochthonous consortium has higher mycorrhizal root colonization of hulless barley by 7-fold and 23-fold in comparison to the non-inoculated controls, under moderate drought and severe drought conditions, respectively. Water stress decreased grain yield and thousand-kernel weight of hulless barley. The reduction was less pronounced in AMF inoculated plants compared to the non-inoculated control ones. Plants with higher mycorrhizal colonization showed higher grain yield and thousand-kernel weight by approximately 90% and 68.2% with AI, and by 106% and 83% with CI, respectively than control plants with lower AMF colonization, especially under severe drought. At the same time, the amount of K, Cu, Fe, Zn and Ca in hulless barley grain increased significantly in AMF inoculated plants with AI as well as with CI. Compared to the control plants, using autochthonous AMF species led to significantly decreased Na content in grain. Fatty acids in hulless barley grain decreased with the severity of water stress. Only under well-watered condition, AMF inoculation enhance C18:0 and C18:1 contents as compared to control plants. Moreover, total polyphenol and flavonoid increased due to AMF inoculation under both medium drought and severe drought conditions. The results obtained herein indicated that inoculation with AMF can enhance the water tolerance resulting in higher hulless barley grain yield and quality. Therefore, using AMF as biofertilizers may be important in regions suffering from lack of water in order to ensure sustainable agricultural systems.