Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic resistant strains. Here, we show that upon exposure to allicin, growth of Escherichia coli is strongly inhibited. Growth inhibition coincides with a significant drop of total sulfhydryl concentrations and induction of the heat shock and oxidative stress response. In contrast to diamide, a potent inducer of disulfide stress, allicin does not induce cystine bond formation in proteins such as cytoplasmically expressed alkaline phosphatase PhoA. We identified and quantified the allicin-induced modification S-allylmercapto-cysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential OxICAT labeling. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Our results indicate that the mode of action of allicin is a combination of disturbance of the redox balance and inactivation of crucial metabolic enzymes.