Plasma-activated water (PAW) is a new technology applied in biomedical research, agriculture, and the food industry. However, its safety and long-term efects require further investigation. Therefore, the current study was designed to evaluate the impact of PAW oral gavage on body weight, food intake, behavior (tail suspension test and open feld test), internal organs relative weight, blood biochemical indicators, intestinal microbiota and liver and kidney histopathology in 10-week-old female mice (n=30). Mice were divided into three groups each containing 10 mice received orally (gastric lavage) either distilled water (DW), or DW exposed to cold atmospheric plasma (CAP) for 3 min (PAW-3), or 15 min (PAW15) a dose of 200 μl/mice (3 times/week) for 4 weeks. Compared with the DW, the results showed that both PAW-3 and PAW-15 signifcantly increased urea concentration (P=0.044). The PAW-3 group showed a signifcantly higher neutrophil/ lymphocyte ratio (P=0.043), and higher lactobacilli count (P=0.002). However, PAW-15 showed signifcantly higher alkaline phosphatase levels (P=0.039), and a signifcantly lower coliform count (P<0.001). No signifcant diferences (P>0.05) in body weight, food intake, organ relative weights, behavior tests, or major biochemical parameters were observed. Liver and kidney tissues in PAW-treated groups, particularly PAW-15, exhibited severe pathological changes, including hepatocellular necrosis, glomerular atrophy, interstitial infammation, and tubular degeneration, indicating substantial organ damage associated with PAW exposure. In conclusion, PAW’s impact on biochemical markers, microbiota, and organ health raises safety concerns, warranting further long-term studies