Petiveria alliacea L. (Phytolacaceae) is a medicinal plant with a broad range of traditional therapeutic properties, including the treatment of dysentery and intestinal infections caused by protozoan parasites. However, its effects against Entamoeba histolytica have not been reported yet. We investigated the antiamoebic activity present in the leaves of P. alliacea Antiamoebic activity was evaluated in methanolic and aqueous extracts, as well as in the hexanic, methanolic, and EtOAc fractions. The P. alliacea methanolic extract showed a better antiamoebic activity than the aqueous extract with an IC50 = 0.51 mg/ml. Likewise, the hexanic fraction was the most effective fraction, showing a dose-dependent activity against E. histolytica, with an IC50 = 0.68 mg/ml. Hexanic subfraction 12-19 showed the highest antiamoebic activity at 0.8 mg/ml, producing 74.3% growth inhibition without any toxicity in mammal cells. A major component in subfraction 12-19 was identified as isoarborinol, which produced 51.4% E. histolytica growth inhibition at 0.05 mg/ml without affecting mammal cells. The P. alliacea leaf extract has antiamoebic activity that can be attributed to a major metabolite known as isoarborinol.