Sortase A (SrtA) plays a crucial role in the attachment of virulence factors characterized by the LPXTG sequence to the peptidoglycans present on the cell wall surface, thereby rendering it a significant target for anti-virulence agents aimed at combating Staphylococcus aureus (S. aureus) infections. This study focuses on the compound 3, 3'- (1,4-phenylene) diacrylaldehyde (DCA), selected as an anti-anchoring agent for covalent inhibitors. To elucidate the targets of DCA in inhibiting S. aureus and to investigate the molecular mechanism underlying the covalent inhibition of SrtA, proteomics and high-resolution mass spectrometry were employed. The findings indicate that compounds containing α, β-unsaturated aldehydes can covalently modify the catalytic residue Cys184 of SrtA. This modification effectively inhibits the transpeptidation function of SrtA, which is responsible for cell wall anchoring, thereby obstructing the attachment of virulence factors such as IgG-SpA, FnbA, ClfA, Clfb, SdrC, Sas, and Can to the cell wall. As a result, this inhibition impedes immune evasion, biofilm formation, adhesion, and subsequent infection by S. aureus. Furthermore, it disrupts the NADH/NAD+ redox homeostasis and diminishes the virulence phenotype by interfering with the glycolytic pathway and the tricarboxylic acid (TCA) cycle of S. aureus. These findings confirm at the molecular level that compounds containing α, β-unsaturated aldehydes possess the potential to serve as anti-virulence drugs that covalently inhibit SrtA.