Recent studies demonstrate that ursolic acid (UA) presents potent antimicrobial activity against a few microorganisms, however, the underlying mechanisms remain elusive. Moreover, little is known about the effect of UA on carbapenem-resistant Enterobacter cloacae (CREC). In this study, we aimed to investigate the mechanisms underlying-antimicrobial activity of UA and its mode of action against CREC. Minimum inhibitory concentrations (MIC) of UA against CREC were determined by the agar dilution method. The antibacterial effect of UA against CREC was elucidated by evaluating changes in intracellular ATP concentration, intracellular pH, membrane potential, membrane integrity, and cell morphology. A crystal violet assay, field emission scanning electron microscopy (FESEM), and confocal laser scanning microscopy (CLSM) were applied to examine the effect of UA on biofilm formation. Furthermore, the inactivation of CREC cells in biofilms was explored by CLSM. Our results showed that UA has a MIC of 0.1 mg/mL against CREC. UA treatment inhibited CREC growth and impaired membrane integrity of CREC cells, as measured by the decrease in intracellular ATP level, pH, and membrane potential, as well as distinctive deformation in cellular morphology. Moreover, UA exerted a significant inhibitory effect on biofilm formation of CREC and inactivated CREC cells within biofilms. These findings reveal that UA has potent antibacterial and antibiofilm activity against CREC and, thus, can be used as a natural food preservative to reduce CREC-related infections. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.