Water scarcity is considered as one of the most limiting factors of cereal productivity in Mediterranean agricultural systems. This study aims at investigating the contribution of arbuscular mycorrhizal fungi (AMF) inoculation in improving the tolerance of hulless barley (Hordeum vulgare ssp. nudum L.) to water stress. The experiment was carried out under semi-controlled conditions. Two AMF inoculants: a mixture of five native AMF species Pacispora franciscana, Funneliformis mosseae, Funneliformis geosporum, Rhizophagus irregularis and Glomus tenebrosum (NI) and a commercial inoculant containing six species of Glomus sp. (CI) were tested under two water regimes: well-watered regime (WW) and drought regime (D). Growth parameters such as plant height (PH) and shoot biomass; mineral nitrogen (N), phosphorus (P), potassium (K), sodium (Na), copper (Cu), iron (Fe) and zinc (Zn) contents; photosynthetic pigments chlorophyll a (chl a), chlorophyll b (chl b) and carotenoid (Car); chlorophyll fluorescence parameter (Fv/Fm); leaf relative water (RWC); and antioxidant enzyme catalase (CAT) and peroxidase (POX) activities were evaluated to investigate the effect of both factors. Water stress affected plant growth of hulless barley. However, application of AMF biofertilizers attenuated this negative effect. Both AMF inoculants NI and CI improved hulless barley growth (the PH by 11.3% and 19.8% and the shoot biomass by 26.1% and 41.3%, respectively) in comparison with their controls under a drought regime. Mineral nutrient N, P, K, Cu and Fe uptakes were significantly higher in AMF-inoculated plants compared with non-inoculated ones. Concerning the photosynthetic activity, a positive effect of AMF was observed as well under well-watered and drought conditions. Under drought regime, the activity of antioxidant enzymes increased in plants inoculated with AMF. These results were positively correlated with mycorrhizal root colonization, which was improved by AMF inoculation. In plants inoculated with NI, mycorrhizal root colonization was 2.3 times higher than in plants inoculated with CI under drought conditions. This finding was confirmed by the increase in AMF biomass assessed using specific phospholipid fatty acid (PLFA) and neutral fatty acid (NLFA) C16:1ω5 biomarkers in the rhizospheric soil of NI-treated plants in comparison with those treated with CI. In summary, the use of AMF could reduce drought damages by improving the physiological and biochemical responses of hulless barley. This study highlighted the potential role of AMF inoculation, in particular with native strains, as an innovative and eco-friendly technology for a sustainable crop growing system in arid and semi-arid areas.