Abstract Plants are very vulnerable to pathogen attacks and environmental stress as they are exposed to harsh environments in natural conditions. However, they have evolved a self-defense system whereby reactive oxygen and nitrogen species (RONS) act as double-edged swords by imposing (at higher concentration) and mitigating (at lower concentration) environmental stress. Cold plasma is emerging as a feasible option to produce a variety of RONS in a controlled manner when amalgamate with water. Cold plasma activated/treated water (PAW) contains a variety of RONS at concentrations, which may help to activate the plant’s defense system components. In the present study, we examine the effect of cold atmospheric-air jet plasma exposure (15 min, 30 min, and 60 min) on the water’s RONS level, as well as the impact of PAW irrigation, (assigned as 15PAW, 30PAW, and 60PAW) on tomato seedlings growth and defense response. We found that PAW irrigation (priming) upregulate seedlings growth, endogenous RONS, defense hormone (salicylic acid and jasmonic acid), and expression of key pathogenesis related (PR) gene. 30 min PAW contains RONS at concentrations which can induce non-toxic signaling. The present study suggests that PAW irrigation can be beneficial for agriculture as it modulates plant growth as well as immune response components.