keratitis (AK) is a rare but serious infection of the eye and can lead to blindness. The effective and safe medical therapy remains unclear for AK until present. Antimicrobial activity and biological characteristic of chitosan encourage screening of it against . Thus, in vitro anti-amoebic activities of commercial chitosan and nano-chitosan were tested on pathogenic genotype T4, a causative agent of human AK. The spp. was isolated from the keratitis patient The genotype T4 was approved using PCR method followed by sequencing technique. Chitosan nanoparticles was prepared using ionic gelation method and characterized by their physicochemical properties. In the present study, the in vitro activity of serial dilutions (12.5, 25, 50, 100, and 200 µL/mL) of commercial chitosan and nano-chitosan were evaluated against trophozoites and cysts. The finding of nano-chitosan particle size by DLS was 118 nm with a PDI of about 0.134. Zeta potential value was found to be 42.7 mV. The obtained results showed that the tested chitosan and nano-chitosan presented anti-amoebic activities dependent to time and concentration. The inhibitory effect of the chitosan and nano-chitosan is enhanced by increasing the concentration and incubation time. The inhibitory effect of nano-chitosan on both trophozoites and cyst was more than chitosan. According to the results, nano-chitosan shows the potent activity against T4 and could be used for the development of novel and safe therapeutic approaches in the future.