The pulsed electric field method, which is cited as a non-thermal physical sterilization method, enables continuous treatment using a simple protocol. Given that this method causes minimal protein denaturation, it can be used to sterilize eggs, fish, and similar protein-rich food items, if its bactericidal strength and energy efficiency can be improved. In this study, the ability of a pulsed electric field to sterilize Vibrio fischeri was examined. When the current applied to V. fischeri was limited, it was impossible to sterilize the bacteria even when the applied voltage and applied time were increased up to 13 kV and 15 min, respectively. Subsequently, when helium flow was used to increase the current to V. fischeri, sterilization was confirmed at an applied voltage of 13 kV for 5 min or more, and ozone water was detected in the bacterial suspension. Here, we show that sterilization could be achieved due to factors besides heat because applying the voltage at 20 °C or lower facilitated sterilization.