This study aimed to investigate the inactivation of Pseudomonas tolaasii (P. tolaasii) strain Pt18 inoculated on button mushroom (Agaricus bisporus) by non-thermal plasma (NTP), using surface dielectric barrier discharge (SDBD). Furthermore, the effects of NTP on the postharvest preservation of Agaricus bisporus during 21 days of storage at 4 ◦C were studied. The so-called response surface methodology (RSM) was employed to investigate the efficacy of the plasma process parameters on bacterial inactivation and qualitative characteristics of Agaricus bisporus. The NTP at the optimum conditions of treatment time of 180 s and excitation frequencies of 6200 Hz under hydrogen peroxide vapor and air gas (H2O2+air) reduced P. tolaasii on Agaricus bisporus by 4.23 log CFU g 1 after 21 days of storage. The corresponding biological properties of NTP treated and control samples were also assessed. The results of firmness and moisture content suggested that plasma treatment could delay mushroom softening. In addition, vitamin C content of the treated button mushrooms increased despite observing insignificance (P > 0.05) in pH. Hydrogen peroxide vapor and argon gas (H2O2+Ar) treatment showed less Agaricus bisporus color changes during 21-day period. Therefore, it can be concluded that NTP as a novel technology has an excellent potential for improving the microbial contamination and enhancing qualitative properties of Agaricus bisporus.