We present the case report of the first identification of Acanthamoeba as a causative agent of keratitis in the Slovak Republic. For the first time, Acanthamoeba sp. Group III was isolated from a 53-year-old patient with keratitis, which was manifested after an injury of the right eye. A delayed visit to a physician as well as a late diagnosis of the illness led to the advanced stage of eye disease. As the treatment with itraconazol and cornea transplantation showed no result, enucleation of the eye was decided. Acanthamoeba ludgunensis was also the causative agent of keratitis in a 39-year-old patient wearing contact lenses. His complaints occurred a month after bathing in a thermal swimming pool. The symptoms presented in the left eye were those of herpetic keratitis, and led to a cloudy cornea with circular infliltrate and poor vision. A prompt clinical and laboratory diagnosis, along with treatment with propamidine-isetionate resulted in a significant improvement of the eye condition. Contact lenses were probably related to another case of Acanthamoeba keratitis. The patient, a 15-year-old girl, kept wearing contact lenses during bathing in various swimming pools and in the sea; her contact lenses were also regularly washed under tap water. Due to the fact that cysts of Acanthamoeba sp. group II were found in the contact lens solution, this is presumed to be the source of the eye infection.