The repurposing of drugs is one of the most competent strategies for discovering new antimicrobial agents. Vildagliptin is a dipeptidyl peptidase-4 inhibitor (DPI-4) that is used effectively in combination with metformin to control blood glucose levels in diabetic patients. This study was designed to evaluate the anti-virulence activities of this combination against one of the most clinically important pathogens, Pseudomonas aeruginosa. The current findings show a significant ability of the vildagliptin–metformin combination to diminish biofilm formation, bacterial motility, and the production of virulent extracellular enzymes and pyocyanin pigment. Furthermore, this drug combination significantly increased the susceptibility of P. aeruginosa to oxidative stress, indicating immunity enhancement in the eradication of bacterial cells. In compliance with the in vitro findings, the histopathological photomicrographs of mice showed a considerable protective effect of the metformin–vildagliptin combination against P. aeruginosa, revealing relief of inflammation due to P. aeruginosa-induced pathogenesis. P. aeruginosa mainly employs quorum sensing (QS) systems to control the production of its huge arsenal of virulence factors. The anti-virulence activities of the metformin–vildagliptin combination can be interrupted by the anti-QS activities of both metformin and vildagliptin, as both exhibited a considerable affinity to QS receptors. Additionally, the metformin–vildagliptin combination significantly downregulated the expression of the main three QS-encoding genes in P. aeruginosa. These findings show the significant anti-virulence activities of metformin–vildagliptin at very low concentrations (10, 1.25 mg/mL, respectively) compared to the concentrations (850, 50 mg/mL, respectively) used to control diabetes.