Glyphosate (GLY), one of the most extensively applied broad-spectrum herbicides worldwide, frequently contaminates soil and aquatic ecosystems, posing serious threats to human health, non-target organisms, soil microbial communities, and environmental sustainability. In the present study, phytoremediation using Medicago sativa (MS) was evaluated for the removal of GLY from contaminated soil under greenhouse conditions, with remediation efficiency enhanced through inoculation with two bacterial bioagents, Bacillus sp. h10 (BS) and Pseudomonas aeruginosa KZFS4 (PA). Biochemical parameters, including superoxide dismutase (SOD), catalase (CAT), hydrogen peroxide (H2O2), and malondialdehyde (MDA), together with detoxification-related gene expression, were investigated in the roots and leaves of MS exposed to GLY stress. The combined application of MS with BS + PA, followed by MS + PA and MS + BS, significantly decreased GLY residues in soil and increased GLY accumulation in plant roots and leaves after 1, 3, 7, and 10 days compared with MS treatment alone. In vitro batch equilibrium experiments demonstrated that BS and PA desorbed 33.63 and 40.56 µg g−1 of GLY, respectively, thereby enhancing its removal from soil. The persistence of GLY was highest in contaminated soil without treatment, exhibiting a half-life (t1/2) of 52.66 days, whereas the shortest half-life (6.69 days) was recorded in soil treated with MS combined with BS and PA relative to sterilized contaminated soil. Furthermore, inoculation with BS and PA markedly increased SOD and CAT activities in MS tissues, while significantly reducing H2O2 and MDA accumulation, indicating alleviation of oxidative stress. GLY exposure also triggered substantial upregulation of detoxification-associated genes, including cytochrome P450, glutathione S-transferases (GST), glycosyltransferases (GTs), and ABC transporters in MS. These findings demonstrate that the integration of BS and PA with phytoremediation effectively accelerates GLY dissipation and reduces pesticide-associated toxicity in contaminated soils and plants