The chemical composition, antitumor activity and toxicity of the essential oil from Lippia microphylla leaves (OEL) were investigated. The major constituents were thymol (46.5%), carvacrol (31.7%), p-cymene (9%), and γ-terpinene (2.9%). To evaluate the toxicity of OEL in non-tumor cells, the hemolytic assay with Swiss mice erythrocytes was performed. The concentration producing 50% hemolysis (HC50) was 300 μg/mL. Sarcoma 180 tumor growth was inhibited in vivo 38% at 50 mg/kg, and 60% at 100 mg/kg, whereas 5-FU at 50 mg/kg caused 86% inhibition. OEL displays moderate gastrointestinal and hematological toxicity along with causing some alteration in liver function and morphology. However, the changes were considered reversible and negligible in comparison to the effects of several anticancer drugs. In summary, OEL displays in vivo antitumor activity and a moderate toxicity, which suggests further pharmacological study.