This study investigated the antibacterial mechanism of action of the seed essential oil of Eleutherococcus senticosus (ESEO) against foodborne pathogenic bacteria. Preliminarily, the ESEO (1000 μg disc−1) showed potential antibacterial effect as diameter of inhibition zones (12.0 ± 0.2–37.0 ± 2.0 mm) against the tested foodborne pathogens. The MIC and MBC values of ESEO against the tested bacteria were found in the range of 125–500 and 500–1000 μg mL−1, respectively. At MIC concentration, the ESEO had potential inhibitory effect on the cell viability of the tested pathogens. In addition, SEM analysis showed the inhibitory effect of ESEO as confirmed by considerable morphological alterations on the cell wall of B. cereus ATCC 13061 and E. coli O157:H7 ATCC 43889. Moreover, the ESEO revealed its mode of action against foodborne pathogens on membrane integrity as confirmed by release of extracellular ATP, 260-nm absorbing materials and leakage of potassium ions. These findings confirm that the ESEO can be used as a potential antibacterial agent in food industry to inhibit the growth of various foodborne pathogens.