The present study aimed to evaluate the effect of Bacillus amyloliquefaciens and/or Arbuscular Mycorrhizal Fungi (AMF) as natural biofertilizers on biomass, yield, and seed nutritive quality of soybean (Giza 111). The conditions investigated include a well-watered (WW) control and irrigation withholding at the seed development stage (R5, after 90 days from sowing) (DS). Co-inoculation with B. amyloliquefaciens and AMF, resulted in the highest plant biomass and yield under WW and DS conditions. The nuclear DNA content analysis suggested that co-inoculation with B. amyloliquefaciens and AMF decreased the inhibition of drought stress on both the size and granularity of seed cells, which were comparable to the normal level. The single or co-inoculation with B. amyloliquefaciens and AMF increased the primary metabolites content and alleviated the drought-induced reduction in soluble sugars, lipids, protein and oil contents. Plant inoculation induced the expression of genes involved in lipid and protein biosynthesis, whereas an opposite trend was observed for genes involved in lipid and protein degradation, supporting the observed increase in lipid and protein content. Plant inoculated with B. amyloliquefaciens showed the highest α-amylase and β-amylase activities, indicating improved osmolyte (soluble sugar) synthesis, particularly under drought. Interestingly, single or co-inoculation further strengthen the positive effect of drought on the antioxidant and osmoprotectant levels, i.e. phenol, flavonoid, glycine betaine contents, and glutathione-S-transferase (GST) activity. As a result of stress release, there was a decrease in the level of stress hormones (abscisic acid, ABA) and an increase in gibberellin (GA), trans-zeatin-riboside (ZR), and indole acetic acid (IAA) in the seeds of inoculated plants. Additionally, the ATP content, hydrolytic activities of plasma membrane H -ATPase, Ca -ATPase, and Mg -ATPase were also increased by the inoculation.