Background & Objectives: Cold Plasma is an emerging non-thermal, chemical-free, environmentally friendly disinfection technology. Plasma-activated water has received considerable attention from researchers in recent years. Despite extensive studies on the antibacterial effects of plasma-activated water, its anti-eukaryotic effects have not been identified. In humans, Acanthamoeba causes granulomatous encephalitis, skin ulcers, and Acanthamoeba keratitis. Considering the health importance of Acanthamoeba, this study investigated the anti-amoeba effect of plasma-activated water on trophozoites and cysts of Acanthamoeba castellanii.