Amoebic keratitis is difficult to treat without total efficacy in some patients because of cysts, which is less susceptible than trophozoites to the usual treatments. We investigated the in vitro effectiveness of the methanolic extract of four Allium species from Turkey against Acanthamoeba castellanii and its cytotoxicity on corneal cells in vitro. Extracts were evaluated for their amoebicidal activities using an inverted light microscope. The effect of the Allium species with concentrations ranging from 1.0 to 32.0 mg/ml on the proliferation of A. castellanii trophozoites and cysts were examined in vitro. For the determination of the cytotoxicity of the Allium species on corneal cells, agar diffusion tests were performed. According to the results obtained from the tests, A. scrodoprosum subsp. rotundum showed remarkable amoebicidal effect on A. castellanii, while the others remained inactive. In the case of cyctotoxic activities, the methanolic extracts of A. scrodoprosum showed no cytotoxicity for the cells in the concentration of 32 mg/ml, while the others exerted significant cytotoxic activities between the concentrations of 1 and 16 mg/ml. As a result, the methanolic extract of A. scrodoprosum could be concluded as a new natural agent against Acanthamoeba. On the other hand, it still needs to be further evaluated by in vivo test systems to confirm the efficiency of its biological effect.