Juglone (5-hydroxy-1,4-naphthoquinone) is a major chemical constituent of Juglans mandshruica Maxim. Recent studies have demonstrated that juglone exhibits anti-cancer, anti-bacterial, anti-viral, and anti-parasitic properties. However, its effect against Acanthamoeba has not been defined yet. The aim of this study was to investigate the effect of juglone on Acanthamoeba. We demonstrate that juglone significantly inhibits the growth of Acanthamoeba castellanii at 3-5 μM concentrations. Juglone increased the production of reactive oxygen species (ROS) and caused cell death of A. castellanii. Inhibition of ROS by antioxidant N-acetyl-l-cysteine (NAC) restored the cell viability. Furthermore, our results show that juglone increased the uptake of mitochondrial specific dye. Collectively, these results indicate that ROS played a significant role in the juglone-induced cell death of Acanthamoeba.