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Aim: The aim of this study was to investigate the mode of action of the lavender essential oil (LV) on antimicrobial activity against multi-drugresistant Escherichia coli J53 R1 when used singly and in combination with piperacillin. Method and Results: In the time-kill analysis, a complete killing of bacteria was observed based on colony counts within 4 h when LV was combined with piperacillin during exposure at determined FIC concentrations. Analysis of the membrane permeabilizing effects of LV on treated cultures through their stability against sodium dodecyl sulphate revealed that the LV played a role in disrupting the bacterial cell membrane. The finding is further supported by scanning electron microscopy analysis and zeta potential measurement. In addition, reduction in light production expression of E. coli [pSB1075] by the LV showed the presence of potential quorum sensing (QS) inhibitors. Conclusions: These results indicated that the LV has the potential to reverse bacterial resistance to piperacillin in E. coli J53 R1. It may operate via two mechanisms: alteration of outer membrane permeability and inhibition of bacterial QS. Significance and Impact of the Study: These findings offer a novel approach to develop a new option of phytopharmaceuticals against multi-drug-resistant E. coli.