A Gram-negative bacteria () was exposed to different concentrations (0, 20, and 40 mg/L) of dimethyl phthalate (DMP) for 8 h, and then Fourier transform infrared spectroscopy (FTIR) analysis, lipopolysaccharide content detection, analysis of fatty acids, calcein release test, proteomics, non-targeted metabolomics, and enzyme activity assays were used to evaluate the toxicological effect of DMP on . The results showed that DMP exposure caused an increase in the unsaturated fatty acid/saturated fatty acid (UFA/SFA) ratio and in the release of lipopolysaccharides (LPSs) from the cell outer membrane (OM) of . Moreover, DMP regulated the abundances of phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG) of and induced dye leakage from an artificial membrane. Additionally, excessive reactive oxygen species (ROS), malondialdehyde (MDA), and changes in antioxidant enzymes (i.e., catalase [CAT] and superoxide dismutase [SOD]) activities, as well as the inhibition of Ca-Mg-ATPase and Na/K-ATPase activities in , which were induced by the DMP. In summary, DMP could disrupt the lipid asymmetry of the outer membrane, increase the fluidity of the cell membrane, and destroy the integrity of the cell membrane of through lipid peroxidation, oxidative stress, and ion imbalance.