Aim To evaluate the antimicrobial effect of paeoniflorin against carbapenem‐resistant Klebsiella pneumoniae (CRKP). Methods and Results Minimum inhibitory concentration (MIC) of paeoniflorin against CRKP was determined by agar dilution method. Changes in intracellular ATP concentration, intracellular pH (pHin), cell membrane potential and membrane integrity were investigated to assess the influence of paeoniflorin on cell membrane damage. Additionally, alterations in cell structure of CRKP cells and cell damage within biofilms were examined. The results indicated that paeoniflorin was effective against CRKP at MIC of 1·2 mg ml−1. Paeoniflorin destroyed the integrity of CRKP cell membrane, as was confirmed by decrease of intracellular ATP, pHin, membrane potential, as well as distinctive alteration in cell morphology, resulting in leakage of CRKP intracellular components. Moreover, paeoniflorin displayed a markedly inhibitory influence on biofilm formation of CRKP and inactivated CRKP cells within biofilms. Conclusions Paeoniflorin shows promise as an effective antibiotic against CRKP with the potential to be an alternative therapeutic agent. Significance and Impact of the Study This study provides information about the potential use of paeoniflorin to reduce the infection of CRKP in clinical practice and food production.