Infections caused by parasites in humans represent one of the main public health concerns. Amoebiasis, a parasitic infection caused by Entamoeba histolytica (E. histolytica), is considered endemic in Mexico, where Argemone mexicana (A. mexicana) has been used in traditional medicine to treat intestinal parasitic diseases. The objective of this work was to evaluate the potential biological activity of A. mexicana on E. histolytica. For this purpose, a methanolic extract was prepared from A. mexicana leaves, and a differential fractionation was carried out with solvents of different polarities. The inhibitory capacities of the extract and its fractions were evaluated in vitro using HM1-IMSS, a strain of Entamoeba histolytica. A. mexicana extract was found to have a growth-inhibiting activity for E. histolytica, showing IC50 = 78.39 μg/mL. The extract was characterized phytochemically, and the methanolic extract fractions were analyzed by liquid chromatography (HPLC) and mass spectrometry (MS). Berberine and jatrorrhizine were present in the active fractions, and these compounds may be responsible for the antiparasitic activity. The identification of amoebicidal activity of A. mexicana on E. histolytica gives support to the traditional use. Further studies with berberine and jatrorrhizine will be carried out to understand the mechanism involved.