Facing climate change, the development of innovative agricultural technologies securing food production becomes increasingly important. Plasma-treated water (PTW) might be a promising tool to enhance drought stress tolerance in plants. Knowledge about the effects of PTW on the physiology of plants, especially on their antioxidative system on a long-term scale, is still scarce. In this work, PTW was applied to barley leaves (Hordeum vulgare cv. Kosmos) and various constituents of the plants’ antioxidative system were analyzed 30 days after treatment. An additional drought stress was performed after foliar PTW application followed by a recovery period to elucidate whether PTW treatment improved stress tolerance. Upon PTW treatment, the Total Antioxidant Capacity (TAC) in leaves and roots was lower in comparison to deionized water treated plants. In contrast, PTW treatment caused a higher content of chlorophyll, quantum yield and total ascorbate content in leaves compared to deionized water treated plants. After additional drought application and subsequent recovery period, an enhancement of values for TAC, contents of malondialdehyde, glutathione as well as activity of ascorbate peroxidase indicated a possible upregulation of antioxidative properties in roots. Hydrogen peroxide and nitric oxide might mediate abiotic stress tolerance and are considered as key components of PTW.