OBJECTIVE: Different Acanthamoeba species are among the most ubiquitous organisms causing serious diseases in humans including central nervous system (CNS) and eye infections. Contact lenses, lens care solutions and the hospital environments particularly the indoor and outdoor environments of ophthalmology wards where people are present with different types of eye diseases, are the potential sources of human infection. The purpose of the present study was the molecular investigation of free-living amoebae in the used contact lenses, lens care solutions and hospital samples from the ophthalmology wards and operating rooms in a referral hospital in southeastern Iran. METHODS: Samples were collected from the lens care solutions, used contact lenses, and from indoor and outdoor environments of the ophthalmology ward and operating room of a major referral hospital in Kerman, Southeastern Iran. The samples were cultured on non-nutrient agar (NNA) in 28-30 RESULT: In total 70 samples were collected from the used contact lenses and lens care solutions, as well as the hospital environment of which 11.4% (8 out of 70) were found positive using NNA culture. Two out of 40 samples (5.0%) from the used contact lenses and the care solutions were found positive for the presence of Acanthamoeba T4 genotype. Acanthamoeba cysts were identified in two out of 22 dust samples (9.1%) collected from the ophthalmology ward and operating rooms. Protacanthamoeba bohemica was found in the soil samples from outdoor environment of the ophthalmology ward. CONCLUSION: This study identified Acanthamoeba species in contact lenses and in indoor environmental samples from the ophthalmology ward. T4 genotype was found on the contact lenses examined in this study. In the dust sampled from within the ophthalmology ward Acanthamoeba sp. was identified.