Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii, with very few therapeutic treatment options. Typically, the choices for treatment are pyrimethamine and sulfadiazine, however their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound oleuropein isolated from Fraxinus rhynchophylla showed anti-T. gondii effects in vitro and in vivo. In Madin-Darby bovine kidney cells, the selectivity of oleuropein was 8.9, which was higher than sulfadiazine and pyrimethamine (3.8 and 2.5, respectively). In infected mice, the inhibition ratio of T. gondii in the peritoneal cavity was 55.4% compared to the negative control group after treatment with 300 mg/kg oleuropein. In addition, inhibitory effects on granuloma, apoptosis, necrosis and cyst-formation were shown in sections of spleen and liver. Oleuropein is therefore a potentially useful anti-T. gondii candidate for clinical application.