Essential oils are increasingly being used in human health and animal farming as alternatives to antibiotics. The aim of this study was to better understand the mode of antimicrobial action of a natural essential oil mix (EO mix) by comparison with the colistin, as an antibiotic. The growth inhibitory concentration (GIC) of the EO mix and colistin was determined by turbidimetry. Escherichia coli exposed to EO mix and colistin were analysed by flow cytometry using the fluorescent dyes 3,3-diethyloxacarbocyanine iodide(DiOC2 (3)) to assess membrane potential, and propidium iodide (PI) and SYTO9 to assess membrane integrity following treatment at the GIC and ½-GIC of the EO mix every h for 4 h. At 1 h, treatment with EO mix and colistin resulted in significant cell membrane alteration and depolarization. Membrane integrity measurements identified four sub-populations that were not distributed in the same way between EO mix and antibiotic treated cells. Colistin at GIC and ½-GIC drastically disintegrated the cells that appeared as debris (69.8% of cells were lysed after 1 h of treatment) whereas the EO mix at GIC altered membrane of a majority of cells (67.4 ± 1.3% of cells were partially altered). Contact with ½-GIC EO mix led to sub-populations that persisted or recovered a physiological state with intact membrane (from 1 h to 4 h of treatment, intact cells increased from 23 to 33%). So, it was demonstrated that the EO mix presented antibacterial action against E. coli. It altered membrane properties by decreasing its polarity and integrity which were reversible phenomena here.