The rise of methicillin-resistant Staphylococcus aureus (MRSA) as a major public health threat underscores a critical need for new antibacterial strategies. Quercetin is a naturally occurring flavonoid with a range of bioactivities, including antibacterial activity against S. aureus. However, how quercetin inhibits S. aureus and binds to its potential molecular target is not well understood. Understanding the interaction of quercetin with potential bacterial targets may provide crucial insights for developing modified derivatives with better drug-like properties. To investigate potential targets of quercetin in S. aureus, we employed a targeted subtractive proteomics approach, which identified the glycosyltransferase MurG as a novel quercetin target. Through rigorous molecular docking and extensive 250 ns molecular dynamics simulations, quercetin was shown to bind stably to MurG, suggesting a mechanism that interferes with the critical peptidoglycan biosynthesis pathway. Molecular Mechanics Generalized Born Surface Area (MM-GBSA) analyses provided quantitative evidence of the complex's stability, indicating a strong and stable interaction with potential therapeutic implications. Principal Component Analysis (PCA) further validated the reduction in MurG's structural flexibility upon quercetin binding, reinforcing the hypothesis that this interaction could effectively inhibit its biological function. The identification of its interaction with MurG provides a foundation for the development of novel, more effective antibacterial agents. This strategy, facilitated by subtractive proteomics, could also be adapted to target other resistant pathogens, demonstrating broad applicability in the fight against antibiotic resistance.