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Background & aim: Ear infections are one of the most common diseases among the clients of ear, nose and throat clinics worldwide. Due to insufficient information about the frequency pattern of the causative species in Kohgiluyeh and Boyerahmad province in southwest of Iran, this study was performed to isolate and identify the bacterial and fungal agents causing the disease in Yasuj. Methods: In the present cross-sectional study, patients with ear infections referred to the ear, pharynx and nose clinic of Shahid Mofatteh of Yasuj during the two-year period of 2016-2017 were sampled by an otolaryngologist. The sample was divided into two parts. Part of the sample was used for direct staining and hot staining (visualization of bacterial and fungal elements) and the second part was cultured in two media of sabouraud's dextrose agar with and without chloramphenicol at least at three points. Phenotypic methods were used to identify bacterial and fungal agents. Data were analyzed using descriptive and analytical tests including one-way ANOVA and chi-square. Results: Of the 341 patients studied, 40% were male and 60% were women aged 5 months to 90 years. The prevalence of fungal and microbial infection was 75% (46.5% fungal agents, 23.7% bacterial agents and 4.8% fungal and bacterial infection respectively) and the duration of the disease varied from two days to 29 years. The largest age range involved 31-40 (27%). 69.8% of patients were housewives and 74.6% had history of manipulating the ear canal with contaminated equipment. Major fungal causes of the disease were in the order of frequency, including Aspergillus niger, Aspergillus flavus, and important bacterial agents including Staphylococcus aureus and Pseudomonas aeruginosa. Concurrent infection with fungal and bacterial agents was observed in 16 (4.8%) patients. Conclusion: Aspergillus niger and Staphylococcus aureus were the most common fungal and bacterial agents that were consistent with the pattern of frequency distribution of factors causing ear infections in Iran. Given that ear manipulation with external devices is one of the most important causes of infection in the present study, providing effective education to citizens can help reduce disease cases. In addition for teaching health tips and treatment, accurate laboratory diagnosis is necessary.