Acanthamoeba keratitis (AK) is a painful, sight-threatening, and difficult-to-treat corneal infection caused by the ubiquitous free-living amoebae Acanthamoeba species. The aim of the present study was to compare the severity of keratitis, caused by Acanthamoeba hatchetii and Acanthamoeba castellanii infections, and to assess the therapeutic effects of combined chlorhexidine (CHX) and NeosporinA (R) treatment in rats. The rats were first divided into two groups, in which the eyes of the animals were infected with A. hatchetii or A. castellanii trophozoites. On day 5, all corneas were examined in order to determine the degree of infection (grade 0 to 3), and animals were divided into two new groups, treatment and infected control groups. The treatment was continued for 28 days, followed by excision and histological evaluation of the corneas. In conclusion, the clinical picture progressed more rapidly and severely in eyes infected by A. castellanii, while it was non-invasive and slower to progress with A. hatchetii. Moreover, eyes infected by A. hatchetii responded quicker and more positively to therapy, consistent with its clinical course, while a longer recovery was seen with A. castellanii. Histological examinations revealed the presence of A. castellanii and A. hatchetii trophozoites in the stroma of eyes of the treatment and control groups. As a result, our findings suggest that a combination of Neosporin with lower doses of CHX may be beneficial to treat patients with early diagnosis of AK.