Background: Hospital operating rooms are one of the most sensitive parts of hospital which observe their hygiene is very important. Fungal spores present in the hospital air can cause many nosocomial infections. The Aim of this study was to determine the prevalence of fungal pollution in the air of operation room in the Alborz Province selected hospitals. Methods: In this study, the operating rooms of two hospitals were selected according to the type of activity and hospitalized patients for sampling. Sampling was carried out before the first operation at 6 AM, when the operating room had been disinfected by UV light, and after the last operation at 12 PM, when the pollution was too much. Samples were cultured on Sabouraud Dextrose Agar according to open plate method. Results: The most fungi found in the hospitals operating rooms air were allocated to the Cladosporium, Penicillium, Mucor, and Aspergillus flavus genus which were equal to 36.8%, 18.47, 13.2, and 10.5%, respectively. The highest and lowest pollution rate was related to operating room and cosmetic surgery room, respectively. Conclusion: According to the obtained results, a positive pressure ventilation system can be a good option to control and prevention of nosocomial infections caused by fungi agents.