Purpose <p>Recent years have seen increased focused on developing new anti-parasitic drugs that are both highly effective and low in toxicity, as the widespread use of existing anti-parasitic drugs has raised growing concerns. Natural products have gained significant interest due to their diverse biological activities with minimal toxic side effects. Our previous studies have already demonstrated the good anti-<i>E. tenella</i> effect of Perillyl Alcohol. To further investigate its efficacy against other protozoa, we selected <i>Toxoplasma gondii</i> as the researchsubject.</p> Methods <p>In this study, we utilized the CCK-8 assay in vitro to assess the cytotoxicity of Perillyl Alcohol on DF-1 cells. The impact of Perillyl Alcohol of <i>T. gondii</i> tachyzoite invasion and intracellular proliferation were investigated in vitro. In vivo, we evaluated the effect of Perillyl Alcohol on the pathogenicity of <i>T. gondii</i>, including host survival time, liver and spleen tissue damage, cysts formed in the brain. Furthermore, we analyzed the expression levels of isoprenylation-related genes using quantitative PCR (qPCR).</p> Results <p>The half maximal inhibitory concentration (CC50) of Perillyl Alcohol against DF-1 cells was determined to be 525.0. In vitro studies showed that treatment with Perillyl Alcohol effectively inhibited the invasion rate and intracellular proliferation of <i>T. gondii</i> tachyzoite. The half maximal inhibitory concentration (IC50) was calculated to be 314.3, and further analysis yielded a selectivity index (SI) of 1.67. In vivo, Perillyl Alcohol treatment prolonged the survival time and increased the survival rate of <i>T. gondii</i>-infected mice, while reducing the parasite burden in liver and spleen tissues. It also demonstrated a certain protective effect against<i> T. gondii</i>-induced tissue damage, including effectively alleviating hepatosplenomegaly and mitigating the elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels induced by hepatic injury. Building on this foundation, we further explored the impact of Perillyl Alcohol on the formation of brain cysts and found that it could significantly reduce the number of brain cysts induced by the Pru strain of <i>T. gondii</i> infection. After treatment with Perillyl Alcohol, the expression levels of isoprenylation-related enzymes <i>Tgmecs</i>, <i>Tgdxr</i>, and <i>Tghdr</i> were significantly reduced.</p> Conclusions <p>These results demonstrate that Perillyl Alcohol may suppress the growth of <i>T. gondii </i>by significantly inhibiting the expression of enzymes involved in isoprenoid biosynthesis.</p>