Abstract: Kiwifruit bacterial canker has been one of the most devastating diseases in global agriculture, due to its highly destructive nature and persistent presence. Pseudomonas syringae pv. actinidiae (PSA) has been identified as the causative agent responsible for the kiwifruit bacterial canker. Among them, cold Plasma (CP) can be expected to partially or fully ionize the agent at ambient temperature. The sources of CP can be the dielectric barrier discharge (DBD), gliding arc discharge, corona discharge plasma jet, and microwave/radio frequency plasma. In interaction with water, CP can be used to induce the plasma-activated water (PAW) at the elevated levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS). This transformation of CP into PAW can be potential for agriculture and biomedicine, due to the significant antimicrobial properties of ROS and RNS content. A systematic exploration was conducted to explore the antimicrobial efficacy and potential mechanism of PAW against PSA, particularly the relationship between discharge time and the bactericidal effect of PAW. Different PAW samples were prepared with the activation times of 0, 30, 60, 90, and 120 s in a generator equipped with DBD. The discharge parameters were given: discharge power 40 W, (input voltage 40 V, current (1.0 ± 0.1) A), frequency 200 Hz, and the air medium with an air gap of 3 mm. The bactericidal mechanism of PAW on PSA was investigated to determine the pH, oxidation reduction potential (ORP), conductivity, and concentrations of three oxidizing agents, including hydrogen peroxide (H₂O₂), ozone, and nitrite ions in the samples of PAW. PSA morphology was examined to assess the DNA integrity, damage to the cell membrane, intracellular ROS accumulation, and leakage of contents using particle size analysis and fluorescent staining. The inactivation mechanism of PSA was investigated by PAW. There was a significant correlation between the activation time and the bactericidal efficacy with a significant (P<0.05) reduction of 4.38 lg CFU/mL after 120 s of PAW treatment, compared with the control. The acidification of sterile deionized water was attributed to H₂O₂, ozone, and nitrite ions during plasma discharge. The conductivity, ORP, and concentration of the three substances were then determined in the PAW. There was a significant increase (P<0.05) with increasing discharge time. In addition, the low pH environment of the PAW was used to maintain its bactericidal efficacy. There was a positive correlation among the DNA damage, membrane permeability barrier disruption, leakage of intracellular substances, and the accumulation of ROS with the PAW activation time. The lethal impact of PAW on PSA was attributed to cellular oxidative damage. The inherent acidic pH was induced to elevate the redox potential in the presence of aqueous active substances, such as ozone, H₂O₂, and nitrite ions in the PAW. The antimicrobial mechanisms of PAW against PSA greatly contributed to the potential applications and prevention of agricultural disease.