Updated project metadata. Allicin from garlic is an antimicrobial substance that is effective against several ESKAPE pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). However, the antimicrobial mode action of allicin has not been revealed in Gram-positive bacteria. Here, we have studied the changes in the transcriptome by Allicin which revealed a thiol-specific oxidative stress response in S. aureus. Using shotgun proteomics, we identified numerous proteins that were modified by protein S-sulfoallylation in S. aureus. Allicin further caused an oxidative shift in the bacillithiol (BSH) redox potential as revealed by Brx-roGFP2 biosensor measurements. In summary, our results revealed that allicin acts via sulfoallylation of Cys residues in proteins causing an impaired redox state in S. aureus.