<jats:p><jats:italic>Toxoplasma gondii</jats:italic> is an obligate intracellular pathogen that infects warm-blooded animals and humans. However, side effects limit toxoplasmosis treatment, and new drugs with high efficiency and low toxicity need to be developed. Natural products found in plants have become a useful source of drugs for toxoplasmosis. In this study, twenty natural compounds were screened for anti-<jats:italic>T. gondii</jats:italic> activity by Giemsa staining or real-time fluorescence quantitative polymerase chain reaction (qPCR) <jats:italic>in vitro</jats:italic>. Among these, licarin-B from nutmeg exhibited excellent anti-<jats:italic>T. gondii</jats:italic> activity, inhibiting <jats:italic>T. gondii</jats:italic> invasion and proliferation in a dose-dependent manner, with an EC<jats:sub>50</jats:sub> of 14.05 ± 3.96 μg/mL. In the <jats:italic>in vivo</jats:italic>, licarin-B treatment significantly reduced the parasite burden in tissues compared to no treatment, protected the 90% infected mice from to death at 50 mg/kg.bw. Flow cytometry analysis suggested a significant reduction in <jats:italic>T. gondii</jats:italic> survival after licarin-B treatment. Ultrastructural changes in <jats:italic>T. gondii</jats:italic> were observed by transmission electron microscopy (TEM), as licarin-B induced mitochondrial swelling and formation of cytoplasmic vacuoles, an autophagosome-like double-membrane structure and extensive clefts around the <jats:italic>T. gondii</jats:italic> nucleus. Furthermore, MitoTracker Red CMXRos, MDC, and DAPI staining showed that licarin-B promoted mitochondrial damage, autophagosome formation, and nuclear disintegration, which were consistent with the TEM observations. Together, these findings indicate that licarin-B is a promising anti-<jats:italic>T. gondii</jats:italic> agent that potentially functions by damaging mitochondria and activating autophagy, leading to <jats:italic>T. gondii</jats:italic> death.</jats:p>