Use of rhizosphere microorganisms provides an alternative or supplement to conventional plant breeding to improve water deficit tolerance of tomato plants. Experiment was carried out to explore the effect of two microbial species, AMF (Rhizophagus irregularis) and Bacillus subtilis, in single and co-application, on growth, colonization, and molecular aspects of tomato plants under drought stress. Co-inoculated plants showed less reduction in growth traits, photosynthetic pigments, colonization rate, and increased compatible solutes like proline which help in sustaining relative water content than non-inoculated plants. Inoculation considerably enhanced proline dehydrogenase activity, and significantly reduced both Δ1-pyrroline-5-carboxylate reductase Δ1-pyrroline-5-carboxylate synthetase activity causing lower proline accumulation in inoculated plants under drought stress. Co-inoculated plants showed obvious upregulation of antioxidant system, thus facilitating amelioration of oxidative stress through exclusion of reactive oxygen species. No inoculation under drought stress upregulated abscisic acid related genes expression but have no effect in plants inoculated either sole or mixed inoculation. Expression of aquaporin genes was upregulated in plants co-inoculated and with AMF alone under normal condition. However the expression of aquaporin genes were decreased or unaffected in plants inoculated with Bacillus subtilis but increased in non-inoculated plants. Co-applied AMF and bacillus subtilis substantially increase drought tolerance by upregulating proline metabolism, antioxidant enzymes and aquaporin genes. Therefore our results suggest that co-inoculation mediated drought tolerance is linked with increased proline accumulation, enhanced antioxidant enzyme activities and differential regulation of ABA biosynthetic and aquaporin genes, which is vital for osmotic adjustment of host plant.