BACKGROUND: The emergence of antibiotic-tolerant staphylococcal strains, such as vancomycin-intermediate (VISA), poses a significant healthcare challenge and complicates treatment regimens. VISA often exhibits mutations in Krebs cycle enzymes, promoting anaerobic metabolism under physiological conditions and reducing susceptibility to antibiotics and innate immune defenses-factors not typically captured in standard susceptibility testing. Building on these findings, we investigated ethylenediaminetetraacetic acid (EDTA), a chelator that enhances reactive oxygen species (ROS) production, as an adjunct to vancomycin for combating VISA infections. METHODS: RNA sequencing analysis compared gene expression of a well-characterized VISA strain (D712) under physiological versus nutrient-rich conditions. Hydrogen peroxide (HO) killing assays were conducted with and without the hydroxyl radical quencher thiourea, while neutrophil killing assays used ROS scavenger butylated hydroxyanisole (BHA). A murine bacteremia model assessed the effects of vancomycin, EDTA, or their combination on VISA. RESULTS: VISA exhibited downregulation of Krebs cycle enzymes and other genes associated with resistance to iron and ROS-mediated killing under physiological conditions. EDTA, alone or with vancomycin, improved HO-mediated killing compared to vancomycin alone, a response counteracted by thiourea. The combination of EDTA and vancomycin enhanced neutrophil killing of VISA more effectively than either treatment alone, an effect negated by BHA. In vivo, EDTA enhanced vancomycin activity against VISA. CONCLUSIONS: EDTA potentiates vancomycin efficacy against VISA in vivo and enhances susceptibility to HO and neutrophil killing. Reduced expression of Krebs cycle enzymes in VISA suggests that EDTA promotes ROS-mediated bacterial killing, targeting a key mechanism by which persistent staphylococci evade host defenses.