Toxoplasmosis, caused by Toxoplasma gondii (Tg), is one of the most prevalent zoonotic diseases worldwide. Currently, safe and efficient therapeutic options for this disease are still being developed, and are urgently needed. Agrimonia pilosa (A. pilosa) is a rosaceous plant, and widely distributed in Asia. The bioactive compounds in A. pilosa Ledeb include flavonoid, triterpenoids, and isocoumarin, and have been reported to show many pharmacological effects including anti-inflammatory, antioxidant, and antibacterial activities.The aim of this study was to determine Anti-protozoal efficacy of Agrimonia pilosa ethanol extract (APE) against Toxoplasma gondii (Tg). In this study, APE was examined and screened for anti-Tg activity. Seventy percent ethanol was used for herbal extraction. For anti-Tg activity screening, in vitro study with APE using tachyzoit of Tg RH strain infected HeLa cells were performed. Also, in vivo anti- Tg study using the mouse infection model was conducted. As the results, the selectivity of APE was 3.35, which was higher than Sulfadiazine selectivity (2.08). Also, we performed in vivo animal study to evaluate the anti-Tg activity of APE with mouse model. As the results, Anti-toxoplasma activity of the APE was as high as sulfadiazine. These results demonstrate that it can be successfully cured against Tg infection with APE. It could be a promising native herb treatment for the prevention of Tg infection.