Background: Melandrii Herba, a medicinal plant, has been used in Korea for treatment of bacterial and fungal infection. However, the safety and toxicity of Melandrii Herba have not yet been established. Therefore, we investigated the acute and subacute toxicity of an ethanolic extract of Melandrii Herba (MHEE) in Crl: CD Sprague Dawley rats and cytotoxicity of MHEE in vitro. Methods: To study acute toxicity, rats were treated with MHEE at single doses of 0, 500, 1000, and 2000 mg/kg administered by oral gavage, and body weight, clinical signs, and mortality were observed after dosing. To study subacute toxicity, rats were treated with MHEE at doses of 0, 500, 1000, and 2000 mg/kg administered once a day by gavage for 4 weeks. We measured clinical signs, mortality, gross pathological findings, body and organ weights, food consumption, serum biochemistry, and conducted hematology and urinalysis. The cytotoxicity of MHEE was assayed by measuring the viability of prostate cell lines including normal prostate stromal WPMY-1, normal prostate epithelial RWPE-1, and benign prostatic hyperplasia epithelial BPH-1 cells at various concentrations of MHEE in vitro. Results: Single oral doses of MHEE caused no significant difference in rat clinical signs, mortality, or body weight. The lethal dose of MHEE was considered to be >2000 mg/kg. Daily oral doses of MHEE for 4 weeks did not result in any significant changes in rat mortality, gross pathological findings, relative organ weights, food consumption, hematology, serum biochemistry, or urinalysis. At MHEE >1000 mg/kg/day, salivation was increased in both male and female rats. However, the salivation caused by the MHEE treatment was not accompanied by pathological changes in body weight or gross pathological findings, and we considered the salivation as a minor symptom. Therefore, no adverse effects were seen at 2000 mg/kg/day or less. MHEE showed no cytotoxic effects on either normal prostate or benign prostatic hyperplasia cell lines. Conclusions: Administration of MHEE in Crl: CD Spradgue Dawley rats is nontoxic and is safe for at least a month.