Background Drought stress is a catastrophic abiotic stressor that impedes the worldwide output of commodities and the development of plants. The Utilizing biological antioxidant stimulators, Arbuscular mycorrhizal fungi (AMF) are one example increased the plants’ ability to withstand the effects of drought. The symbiotic response of soybean (Glycine max L.) to AMF inoculation was assessed in the experiment presented herewith at different watering regimes (field capacity of 25, 50, and 90%). The vegetative, physio-biochemical traits, and regulation of genes involved in polyamine synthesis in G. max plants were evaluated. Results The results obtained suggested that AMF inoculation has an advantage over plants that were non-inoculated in terms of their growth and all assessed criteria, which responded to drought stress by showing slower development. It is evident that the gas exchange parameters of the soybean plant were substantially reduced by 36.79 (photosynthetic rate; A), 60.59 (transpiration rate; E), and 53.50% (stomatal conductance gs), respectively, under severe stress of drought in comparison to control; non-stressed treatment. However, the AMF inoculation resulted in a 40.87, 29.89, and 33.65% increase in A, E, and gs levels, respectively, in extremely drought-stressful circumstances, when in contrast to non-AMF one that was grown under well-watered conditions. The drought level was inversely proportional to mycorrhizal colonization. The total antioxidant capacity, protein, and proline contents were all enhanced by AMF inoculation, while the malondialdehyde and hydrogen peroxide contents were decreased. Polyamine biosynthesis genes expression; Ornithine decarboxylase (ODC2), Spermidine synthase (SPDS) and Spermine synthase (SpS) were upregulated in drought and to even higher level in AMF’s mild drought inoculated plants’ shoots. This implies that AMF plays apart in the enhanced survival of soybean plants stressed by drought and reduced plant membranes damage by limiting the excessive production of oxidative stress generators; ROS. Conclusions In summary, the present investigation demonstrates that inoculation of AMF may be a supportable and environmentally advantageous method for improving the physio-biochemical traits, plant growth, and polyamine biosynthesis genes of soybean plants in the incident of limited water availability.