Incidences of Acanthamoeba granulomatous encephalitis (AGE) have been increased due to a rise in the number of high-risk people, such as immunodeficient patients. Indeed, immunosuppress situation can render the patient in acquiring opportunistic Acanthamoeba infections. In this study, analysis was carried out to verify the presence of free-living amoebae of Acanthamoeba genus in nasal swabs of cancer patients in hospitals of Tehran, Iran. Detection of isolates was based on morphotyping and PCR sequencing of the Diagnostic Fragment 3 (DF3) to identify strains at the genotype level. In addition, the pathogenic potential of the isolates was assayed using temperature and osmotolerance assays. The obtained results revealed that nine isolated strains belonging to T4 genotype-exhibited pathogenic potential. After sequencing, genotype T4 was found to be the most common one in the samples included in this study. Genotype T3 and T5 were also identified. To the best of our knowledge, this is the first study on the typing of Acanthamoeba strains at the genotype level in cancer patients in Iran and worldwide.