Free-living protozoa of the genus Acanthamoeba can cause one of the most severe, potentially sight-threatening infections of the eye, the so-called A. keratitis. A. keratitis is difficult to treat because, under adverse conditions, the amoeba encyst and medical therapy is often less effective against cysts than against trophozoites. The aim of this study was to investigate evaluate the in vitro effect of the nonpolar subfraction of the methanol extract of garlic (Allium sativum) on the growth of A. castellanii trophozoites and cysts and also its cytotoxicity on corneal cells in vitro. Extract was evaluated for its amoebicidal activity, using an inverted light microscope. The effect of the nonpolar extract with the concentrations, ranging from 0.78 to 62.5 mg/mL on the proliferation of A. castellanii trophozoites and cysts, were examined in vitro. For the determination of cytotoxicity of the extract on corneal cells, agar diffusion tests were performed. The present study demonstrates the in vitro effectiveness of the garlic against the A. castellanii growth curve. Evaluations revealed that garlic inhibits trophozoite growth in dose- and time-dependent ways. In the case of the cyctotoxic acitivities, it showed no cytotoxicity for the cornea cells in the concentration of 3.90 mg/mL. These findings indicate that nonpolar subfraction of the methanol extracts of garlic has amoebicidal, as well as its cysticidal, properties on Acanthamoeba trophozoites and cysts. Garlic alone, and in combination with other amoebicidal agents, may be used in clinical practices after further investigations.