Anacardium occidentale L. leaf demonstrates sexual enhancement effect. Therefore, it can be used as the potential supplement and functional ingredient. However, the ethanolic leaf extract of this plant is a modified form of traditional application and the toxicity evaluation is required. To assess cytotoxicity of the extract, RAW 264.7 cells were treated with A. occidentale leaf extract in the concentration range between 0.625 and 10 mg/mL. Our results showed that the extract showed more than 90% cell viability at the concentration of 2.5 mg/mL after 24-hour exposure. To assure the consumption safety, the acute and subchronic toxicity must be studied. Acute toxicity showed that the extract is safe even at the highest dose of 2 g/kg in both sexes of Wistar rats. No changes in behavior, physiology, gross pathology, and histology were observed. To determine the subchronic toxicity of extract, both sexes of Wistar rats were orally given the extract at doses of 20, 100, and 500 mg/kg once daily for 90 days. No changes in body weight, food, and water intake, motor coordination, behavior, and mental alertness were observed. The significant reduction of white blood cell, platelet, and cholesterol together with increase in MCHC was observed in male rats. The reductions of white blood cell and platelet together with the elevations of hemoglobin and hematocrit were also observed in female rats. However, all changes were in normal range. The current results revealed that an ethanolic extract of A. occidentale leaf was well tolerated via oral consumption up to dose of 500 mg/kg BW for 90 days and did not produce any toxicity. Our in vitro cytotoxicity test also confirmed this safety.