Selected fractions of ethanolic extracts obtained from leaves and roots of Eryngium planum (Apiaceae) were evaluated in vitro for amebicidal activity against Acanthamoeba castellanii. This free-living ameba is the cause of Acanthamoeba keratitis, which is a painful, vision-impairing disease of the eyes, and chronic granulomatous amebic encephalitis. Treatment is very difficult and not always effective because of encystation, which makes the amebae highly resistant to anti-amebic drugs. The search for novel natural amebicidal agents is still of current interest. Fractions of E. planum ethanolic extract from basal leaves: flavonoid fraction (Lf), flavonoid-saponin fraction (Lf-s), saponin fraction (Ls) and phenolic acids fraction (La) and from roots: saponin fraction (Rs) and phenolic acids fraction (Ra) were assayed for antiamebic activity. In the presence of the saponin fractions and phenolic acid fractions (ranging from 1-5 mg/mL), the number of the trofozoites of Acanthamoeba castellanii viable strain 309 decreased during the experimental period (0-72 h). On the other hand, the flavonoid fraction from leaves showed a stimulating activity on the amebae. Almost all fractions (except the flavonoid fraction) showed a time- and dose-dependent amebistatic activity on the trophozoites. Of the fractions tested, the phenolic acid fraction from roots at the concentration of 5 mg/L showed the amebicidal activity on the trophozoites.