Background: Fungi have a worldwide distribution which can cause a broad spectrum of disease ranging from allergic to systemic infections, particularly in immuno-compromised individuals. Fungal spores are an important group of bioaerosols in hospital environment which are an emerging cause of hospital-acquired infection. Nosocomial infections cause significant morbidity and mortality as well as large financial burden on the healthcare system. This study aimed to evaluate the frequency and species distribution of airborne fungi in selected wards of two tertiary hospitals in Tehran, Iran. Methods: In this cross-sectional study, samples were collected during six months from July 2016 to December 2016 by using of settle plate method. Samples were collected from selected wards of Imam Khomeini Hospital and Children's Medical Center and then incubated at 28 °C for 8-10 days. Fungal isolates were identified using the macroscopic features of colony and microscopic characteristics in slide cultures. Yeast isolates were identified by CHROMagar candida medium. PCR-sequencing of ITS1-5.8 S-ITS2 region of ribosomal DNA was used for identification of unknown isolates. Results: A total of 202 colonies including 133 colonies from the Imam Khomeini Hospital and 69 colonies from the Children's Medical Center were isolated. Cladosporium spp. were the most common obtained fungi accounted for 30.1% and 47.8% of all isolates in the Imam Khomeini Hospital and the Children's Medical Center, respectively. Penicillium spp. and Aspergillus spp. were other frequent species in two hospitals. Infectious diseases ward in Imam Khomeini hospital and emergency and urology wards in Children's Medical Center had the highest rate of contamination. Conclusion: According to the results of this study, the frequency and diversity of fungal spores in hospital wards were different. In addition, since the fungal contamination in the hospital environment are affected by various environmental factors and the efficiency of ventilation systems, some of these wards require better ventilation system as well as regular monitoring to remove these fungal bioaerosols in order to maintain the health of patients and health care workers.