The crude extract and hexane, dichloromethane, and methanol fractions obtained from the aerial parts of Pterocaulon polystachyum (Asteraceae) were assayed against Acanthamoeba castellanii, a free-living ameba that causes acute amebic keratitis. Because of its capacity to form cysts, some strains of this protozoan are excellent opportunists and therapy resistant, necessitating a search for new drugs in order to develop more dynamic therapies that make it easier for patients to maintain long-term treatment. In this context, plants with medicinal properties have been analyzed. The broad-spectrum activity against a range of pathogenic fungi shown by extracts of P. polystachyum, together with the use of antifungal drugs as antiprotozoals, made it important to evaluate the amebicidal activity of these plant extracts against A. castellanii. The greatest activity was observed in the treatment with the hexane fraction, which lysed approximately 66% and 70% of the trophozoites in 48 and 72 h, respectively, preventing encystment.