Acanthamoeba species are free living amoeba found widely all over the world. They are responsible for Acanthamoeba keratitis (AK), an infection which is especially seen in contact lens users and after minor corneal traumas, that may lead blindness. At present, antifungals and antiseptics are used for the treatment of AK cases, however, some problems such as long treatment periods and the occurrence of side effects, resistance of cyst forms against drugs, emphasize the need for new drugs. There are some published studies that pointed out the effectiveness of plant extracts and essential oils on Acanthamoeba spp. The aim of this study was to investigate the in vitro effects of essential oils of Mentha x piperita L. (peppermint), Melissa officinalis L. (lemon balm) and Ocimum basilicum L. (basil) belonging to Lamiaceae family, on the cysts and trophozoites of Acanthamoeba castellanii. The strain used in our study, namely A. castellanii T4 genotype, is the most frequently isolated amoeba from environment and also the causative agent of AK and granulomatous amebic encephalitis. For the determination of amebicidal activity, essential oils obtained from Mentha x priperita L., Melissa officinalis L. and Ocimum basilicum L. by Neo-Clevenger type of distillation apparatus have been used. In vitro experiments were performed by using 96-well microplates. Cyst and trophozoite solutions were added on the essential oil dilutions to obtain the last concentrations of 40, 20, 10, 5, 2.5 and 1.25 µg/ml for the cysts, and 10, 5, 2.5, 1.25, 0.625 and 0.313 µg/ml for the trophozoites. After the incubation of microplates at 30oC for 1, 6, 24, 48 and 72 hours, the viability of parasitic forms were evaluated under the light microscope followed by staining trypan blue. It was found that, each essential oil showed amebicidal effect on A.castellani cysts and trophozoites dependent on dosage and time, when compared with the control group, The maximum lethal effect occured with Melissa officinalis followed by Mentha x piperita and Ocimum basilicum, respectively. In our study, susceptibility of A.castellanii trophozoites to essential oils were more than the cysts, as expected. The essential oils of Melissa officinalis and Mentha x piperita showed 100% lethal effect at their highest concentrations whereas the essential oil of Ocimum basilicum showed only 63.3% lethal effect on cysts after 72 hours at the highest concentration (40 µg/mL). The results of this first study investigating the activities of essential oils extracted from Mentha x piperita, Melissa officinalis and Ocimum basilicum against Acanthamoeba spp. cysts and trophozoites, have suggested that, these essential oils could be potential novel and alternative natural products for the treatment of Acanthamoeba spp. infections.