It has been shown that Listeria monocytogenes produces acetoin from glucose under aerobic conditions. A defined medium with glucose as the sole carbon source was used in an aerobic shake flask culture to reliably produce acetoin. Acetoin, the reactive compound in the Voges–Proskauer test, was assayable in the medium and was used to quantify the metabolic response when inhibitors were added to the medium. Inhibitors such as lactic, acetic, propionic and benzoic acids were used to demonstrate the utility of acetoin production as an indicator of metabolic disruption. With increasing levels of inhibitor, the metabolic and growth responses were measured by acetoin production and optical density change, respectively. Both measurements decreased in a similar manner with increasing inhibitor concentrations. The data also showed the apparent mode of action of the inhibitors. A bacteriostatic effect was observed for the protonated organic acids, acetic (4 mmol l−1) and propionic (4 mmol l−1), whereas protonated lactic (4 mmol l−1) and benzoic (0·16 mmol l−1) acids gave an irreversible (apparent bacteriocidal) effect. Lactic, acetic, and propionic acids showed stimulation of metabolic activity at low concentrations, but benzoic did not. Acetoin production is a novel method for quantifying and assessing the mode of action of inhibitors against L. monocytogenes. This system can be used to screen inhibitors for applications in food safety.