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Atmospheric pressure cold plasmas are widely used in the biomedical field as evidenced by developments in recent years. In this study, a quartz tube-guided gas–liquid two-phase discharge has been configured with a hollow needle-water structure. The power source for the device was sinusoidal, and switching between a gas–liquid miscible discharge and a pure gaseous discharge was simple. It was discovered that the gas–liquid discharge was beneficial in terms of reactant species generation and reaction efficiency in terms of processing water. The types of particles, the physicochemical properties, and the concentrations of reactive oxygen and nitrogen species in the discharge were studied spectroscopically. The discharge was discovered to have high levels of H2O2 and NO2−, and the activated water was proven to be effective at sterilizing samples that were contaminated with micro-organisms. Key experimental parameters including the driving voltage and the air flow rate were optimized to achieve the best sterilization conditions. The membrane potential changes in the treated bacteria were also studied to explore the causes of bacterial inactivation. The results showed that the device exhibited a strong bactericidal effect for the gas–liquid mixed phase discharge operating at 17 kV for 5 min with a gas flow rate of 0.3 SLM. In addition, the present device offers enhanced sterilization efficiency relative to the efficiency of conventional plasma sterilization equipment and, therefore, has a wide range of applications. [ABSTRACT FROM AUTHOR]