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Background: Novel antimicrobials with new mechanisms of action are critical to circumvent emerging antimicrobial resistant microorganisms (AMR), such as vancomycin resistant Enterococcus faecium (VanREF). Previous research demonstrated, with transcriptomic analysis and phenotypic assays, that the essential oil components (EOCs) carvacrol (CARV) (1.98 mM) and cuminaldehyde (CA) (4.20 mM) with the antibiotic vancomycin (Van) (0.022 µM), restored the susceptibility of VanREF. This finding suggested that an envelope damage has occurred. Several transport channel-related genes were also differentially expressed including bcr, ecfA1, ecsA-1, yloB and nhaC_2, indicating they could contribute to the mechanism of action of the triple combination CARV-CA-Van. Purpose: The aim of this study was to elucidate the role of transport channels in the antimicrobial mechanism of action of CARV and CA with Van. Methods: The expression levels of bcr, ecfA1, ecsA-1, yloB and nhaC_2 were established using qPCR under the effect of the triple combination, and in the presence of 1 mM calcium or 0.1 mM EDTA (channel blocker) over time. Results: Significant (p ≤ 0.05) changes in expression of yloB and bcr genes were observed at 10 and 30 min, and of ecfA1, ecsA-1 and nhaC_2 at later time points (120 and 360 min). Adding Ca2+ to the combination induced significant changes in the expression of yloB (from -5.67 to -50-fold, +3.68 to -4.4-fold, -1.8 to +6.6-fold and +1.32 to +3.08-fold at 10, 30, 60 and 120 min, respectively). Instead, the addition of EDTA significantly changed the expression of bcr (from -13.5 to -1.11-fold, +15.3 to -1.06-fold, +15.71 to -10-fold at 10, 120 and 360 min, respectively). Since that Ca2+ participates in the stability of bacterial cell wall and EDTA blocks efflux pumps, the current findings suggest the involvement of the efflux system in the mechanism of action of the three-drug combination. Conclusion: Overall, the findings of this study suggest the involvement of transportation channels in the mechanism of action of the CARV-CA-Van combination against VanREF.