Diacetyl (2,3-butanedione) is a key contributor to unpleasant odors emanating from the axillae, feet, and head regions. To investigate the mechanism of diacetyl generation on human skin, resident skin bacteria were tested for the ability to produce diacetyl via metabolism of the main organic acids contained in human sweat. L-lactate metabolism by Staphylococcus aureus and Staphylococcus epidermidis produced the highest amounts of diacetyl, as measured by high-performance liquid chromatography. Glycyrrhiza glabra root extract (GGR) and α-tocopheryl-L-ascorbate-2-O-phosphate diester potassium salt (EPC-K1), a phosphate diester of α-tocopherol and ascorbic acid, effectively inhibited diacetyl formation without bactericidal effects. Moreover, a metabolic flux analysis revealed that GGR and EPC-K1 suppressed diacetyl formation by inhibiting extracellular bacterial conversion of L-lactate to pyruvate or by altering intracellular metabolic flow into the citrate cycle, respectively, highlighting fundamentally distinct mechanisms by GGR and EPC-K1 to suppress diacetyl formation. These results provide new insight into diacetyl metabolism by human skin bacteria and identify a regulatory mechanism of diacetyl formation that can facilitate the development of effective deodorant agents.