Flood events are likely to increase in the near future and are one of the events most threatening to agricultural production. Barley is the fourth most important crop and the cereal most sensitive to excessive moisture stress. Improving the stress tolerance of crops to a variety of stress factors caused by a more volatile climate is an important task for the coming years. The aim of this study was to investigate the efficacy of cold atmospheric plasma-treated water (PTW) as a foliar spray to stimulate the antioxidant system of Hordeum vulgare and thereby improve plant stress tolerance. For this purpose, we analysed the components of the ascorbate-glutathione cycle in barley leaves and roots without stress and under waterlogging conditions. PTW increased the content of total and reduced ascorbate in leaves as well as the content of reduced ascorbate in roots four weeks after treatment. This was observed both, under stress free conditions and after waterlogging and re-aeration. In leaves, enzyme activities also increased after re-aeration, and in roots, total and reduced glutathione levels increased after waterlogging compared to the control. The accumulation of low molecular weight antioxidants may increase tolerance to a variety of stress factors through more efficient scavenging of ROS. Overall, treatment of plants with PTW may trigger an adaptive response that leads to mitigation of the negative effects of stress, and thus, could be used as a priming agent for protection against subsequent more severe stress factors that occur in the plants' natural environment.