Acanthamoeba genus includes pathogenic species which are causal agents of a severe sight-threatening infection of the eye known as Acanthamoeba keratitis (AK). Furthermore, the number of AK is worryingly increasing worldwide, mostly in contact lens users. Until present, there is a general failure to reach a fully effective treatment against AK which is mainly due to the amoebic double-walled cyst stage which forms a protective barrier against drugs. Therefore, drug discovery research towards AK treatment is a must. In this study, Ziziphus vulgaris, a native plant of Asian countries, was checked for its activity against Acanthamoeba. For this purpose and in order to determine the in vitro amoebicidal effects of Ziziphus vulgaris aqueous extract and its fractions (chloroformic, remaining aqueous and primary alcoholic) against Acanthmoeba trophozoites and cysts, activity and sensitivity assays were performed. Moreover, the toxic effect of the extract and its fractions was also tested on murine peritoneal macrophages using a colorimetric tetrazolium salt (MTT) test. The obtained results showed that the chloroformic fraction presented a higher anti-Acanthamoeba activity when compared to the other fractions (Trophozoites/cysts were eliminated, when incubated in a concentration of 50 mg/ml of the fraction, after 24 hours). The calculated active concentrations against Acanthamoeba of these extracts did not shown any high cytotoxicity levels.This study suggests that the Ziziphus vulgaris chloroformic fraction, may present compounds with relevance for the development of novel antiAcanthamoeba drugs.