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To better understand the synergistic antibacterial activity between piperacillin and Lavandula angustifoliaessential oil (LEO) against multidrug-resistant Escherichia coli, we performed microarray transcriptomic analysis of LEO when used alone and in combination with piperacillin against the non-treated control. In total, 90 genes were differentially expressed after the combination of LEO and piperacillin treatment. Among the up-regulated genes, nfsB, nemA, fruA, nfsB, nemAare known to control microbial metabolism and nitrotoluene degradation, which were observed only in the LEO–piperacillin combinatory treatment. Four candidate genes from the microarray result, srIA, srID, waaR and nfsB,were validated by qRT-PCR as these genes showed differential expression consistently in the two methods. Biochemical pathway analysis showed that there was upregulation of genes involved in several biological processes including fructose and mannose metabolism, phosphotransferase system (PTS), lipopolysaccharide biosynthesis and nitrotoluene degradation. Genes involved in microbial metabolism in diverse environments were found both up- and down-regulated in LEO–piperacillin combinatory treatment. Our study provides new information concerning the transcriptional changes that occur during the LEO and piperacillin interaction against the multidrug-resistant bacteria and contributes to unravel the mechanisms underlying this synergism.