The present study was conducted to determine the occurrence of potentially pathogenic free-living amoeba in ophthalmology wards in reference hospitals in Iran. Since an increasing number of Acanthamoeba Keratitis cases after eye surgery and eye trauma have been recently observed in this country, it could be possible that the disinfection procedures undertaken in the clinical setting may not have a good hygiene and disinfection procedures, hence the aim of this study. Therefore, 42 dust and biofilm samples were collected from different areas of ophthalmology wards and checked for the presence of FLA using morphological criteria, PCR based analysis and DNA sequencing. Of the 42 samples from dust and biofilm sources, 18(42.86%) isolates were found to contain FLA and 12(92.3%) isolates belonged to Acanthamoeba T4 genotype. Isolation of the pathogenic genotype T4 from medical instruments, including slit lamp in corneal wards, may be a threat for patients undergoing eye surgery in these wards. Other FLA isolated in this study included Acanthamoeba genotype T5, Vahlkampfia sp, Naegleria australiensis, Vermamoeba vermiformis and Echinamoeba exudans. To our knowledge, this is the first report of the presence of potentially pathogenic FLA in ophthalmology wards in Iran. Improved disinfection methods and monitoring of hospitals ward are thus necessary in this area in order to minimize the risk of infection in patients.