The current research is conducted to investigate the effect of application of mycorrhiza treated soil under water deficit stress on some morphophysiological and biochemical traits of stevia plants obtained from tissue culture. The experiment was done in a greenhouse condition with treatments of two mycorrhiza species at four levels (M1: without mycorrhiza, M2: Glomus mosseae, M3: Glomus intraradices, M4: mixed of both species) and application of water deficit. The irrigation was applied at four levels as follows: I1: maintaining soil moisture at 100% of field capacity, I2: irrigation after reaching 75% of field capacity, I3: irrigation after reaching 50% of field capacity, I4: irrigation after reaching 25% of field capacity. The statistical model was factorial experiment using completely randomized design with 6 replications. Application of mycorrhiza species in water deficit stress conditions dued to 1.70, 4.11, 1.73 and 4.32 fold increase in leaf greenness index, total plant dry weight, main stem height and leaf area parameters, respectively. In addition, these treatments caused to 1.66, 6.13, 29.6 and 1.30 fold increase of potassium, glucose, fructose and total soluble sugars in stevia plants. Under moderate and severe water deficit stress, the use of a mixture of mycorrhiza species in treatments M4I4 and M4I3 compared to treatments M1I4 and M1I3 (without mycorrhiza fungus) increased more than threefold the leaf area of stevia tissue culture plants that positive root symbiosis stevia plants were well clarified by mixing two species of mycorrhizal fungi under water deficit stress and irrigation restrictions.