The deactivation effects and mechanism of plasma‐activated water (PAW) against Staphylococcus aureus biofilm and the inhibitory effect on the biofilm regrowth capacity for the surviving S. aureus post PAW treatment were investigated in vitro. Systematic measurements on bacterial cultivability, metabolic capacity, membrane integrity and intracellular reactive oxygen species (ROS) concentration under two different experimental categories were carried out after PAW treatment. Considerable deactivation effects were discovered on biofilm S. aureus on both prolonging the PAW inducement or treatment time. The rising concentration of intracellular ROS and the PAW‐contained RS might exert synergistic effects in deactivating the biofilm S. aureus. Moreover, the biomass, bacterial cultivability and metabolic capacity of the regrown S. aureus biofilm all significantly declined along with the increasing PAW inducement or treatment time. Therefore, except for the short‐term rapid inactivation effects on the biofilm, the PAW treatment could also exert long‐term inhibitory effects on the regeneration of S. aureus biofilm.

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