Abstract: Cold atmospheric plasma jet is widely used in many fields due to the reactive oxygen species and low temperature for heat-sensitive products. This paper presents the inactivation of bacteria via a pulsed plasma jet with He/O₂ mixed gas. To evaluate the disinfection performance, Staphylococcus aureus was used as an indicator bacteria for experiments. When the plasma jet dealt with agar plates spraying bacteria, it was found that mixed gas has a better performance than pure inert gas, indicated by the disinfection area. The increment of oxygen gas addition was beneficial to the disinfection ability of the plasma jet, while the gas had an opposite effect on the length of jet production. The experiments showed the efficacy of Staphylococcus aureus disinfection could reach up to 99.47% via a helium/oxygen (2%) plasma jet.