Bactericidal and bacteriostatic efficacy of light irradiation, e.g. intense pulsed light (IPL), is mainly derived from the disrupting effects of photoenergy on bacterial biomolecules, including genes, proteins, and metabolites. Previous studies have revealed the effects of IPL on genes and proteins. However, little is known about the influences of IPL on bacterial metabolism. In this study, E. coli K-12 was treated by the IPL for 0-20 s. Time- and dose-dependent decrease of colony-forming units and morphological changes were observed. The IPL-induced changes in the bacterial metabolome were characterized by the liquid chromatography-mass spectrometry-based metabolomic analysis of membrane and cytoplasmic metabolites. The results from multivariate modeling and marker identification showed that the metabolites in electron transport chain (ETC), redox response, glycolysis, amino acid and nucleotide metabolism were selectively affected by the IPL treatments. Further examinations of the time course, scale, and biochemical functions of these metabolic changes suggested that the degradation of membrane-bound quinones in the ETC can contribute to many observed changes, including oxidative stress, disruption of intermediary metabolism, nucleotide degradation, and morphological changes. Therefore, the decreases of membrane quinones, especially the depletion of menaquinone-8, is a prominent and triggering event in IPL-elicited bactericidal and bacteriostatic effect in E. coli.