Nonthermal plasma-treated water (PTW) has emerged as a promising area of research, particularly in the food sector, where it shows potential for fresh food sanitation via wash-water processes. When compared to commonly used strong oxidizing sanitizers, only compressed or ambient air and energy are required to generate plasma processed air (PPA), which produces PTW if combined with process or tap water. This excluded hazardous chemical storage and allows dosage and processing on demand. Ozonated water generation would be a comparable process; however, ozone cannot be cycled for reuse because of its instability. Despite the antimicrobial potential of PTW for sanitation, there are only a few publications on its toxicological aspects in the processing of fresh foods. However, this data is urgently needed to drive customer and industry acceptance as well as to address legal application requests. Our study aimed to assess the potential toxic effects of PTW used within an industrial washing process of ready-to-eat (RTE) lettuce (mix of endive, frisée and radicchio) for food hygienizsation including cytotoxicity (XTT-assay with cell lines Caco-2, V79-4, and L929), mutagenicity (Ames-test), and genotoxicity (comet and micronucleus assays). PTW was prepared within 3.5 h (200 L) from tap water using a microwave discharge until reaching a final pH value below 2. The fresh-cut lettuce was washed either with regular process water or with process water supplemented with PTW, both under standard industrial conditions. After washing, no toxicological effects were detected in lettuce washed with or without the addition of PTW in the process water when compared to unwashed fresh-cut lettuce. The results of the presented study demonstrated that PTW supplementation of process water could be used as a potential sanitation agent.