Piriformospora indica and Azospirillum spp. due to great ability to effectively improve plant growth and stress tolerance, have been considered significantly in recent decades. The present study was conducted as factorial based on a randomized complete block with three replications in the research field of Plant Production Engineering and Genetics, Faculty of Agriculture, Maragheh University. The experimental treatments included three levels of stress (full irrigation, irrigation at 70% of field capacity, and irrigation at 50% of field capacity), endophytic fungus Piriformospora indica (non-use and use) and bacterium Azospirillum (non-use and use). The results of the present study showed that the interaction between drought stress and using fungi and bacteria had a significant effect at the probability level of 1% on leaf area, chlorophyll a, the activity of glutathione reductase, ascorbate peroxidase, and catalase; and the content of malondialdehyde, hydrogen peroxide, superoxide dismutase, Fe-SOD isozymes, Mn-SOD, and proline at the probability level of 5% on Cu/Zn-SOD isozyme. The simple effects of the studied treatments were also significant on the parameters of chlorophyll b and carotenoids at the statistical level of 1%. The highest content of superoxide dismutase, Cu/Zn-SOD, Fe-SOD, Mn-SOD isozymes, ascorbate peroxidase, catalase peroxidase and glutathione reductase was obtained from inoculation with bacteria and fungi in irrigation treatment at 50% of field capacity resulting in a reduction by 47.01 and 33.93% in the content of hydrogen peroxide and malondialdehyde in irrigation treatment at 50% of field capacity and the combined use of fungi and bacteria compared to the non-use of fungi and bacteria in the same treatment. So, it can be concluded that the combined use of Piriformospora indica and Azospirillum spp. under conditions of severe drought stress can play an important role in modulating Barley growth.