Acanthamoeba species are an important cause of microbial keratitis that may cause severe ocular inflammation and visual loss. Acanthamoeba 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 here the in vitro amoebicidal activity of methanolic extract of Thymus sipyleus subsp. sipyleus var. sipyleus from Turkish flora against Acanthamoeba castellanii 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 polar extract with the 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 cytotoxicity of the extract on corneal cells, agar diffusion tests were performed. According to results obtained from the tests, the extract evaluated here showed remarkable amoebicidal effect on A. castellanii. In the case of the cytotoxic activities, it showed no cytotoxicity for corneal cells in the concentration of 32 mg/ml. As a result, polar subfraction of the methanolic extract of Thymus sipyleus subsp. sipyleus var. sipyleus could be concluded as a new natural agent for the treatment of Acanthamoeba infections. On the other hand, it still needs to be further evaluated by in vivo test systems to confirm the efficiency of its biological effect.