Background: Otomycosis is defined as a superficial fungal infection, accounting for about 10% of infectious otitis externa cases. Objectives: This study investigated patients with suspicious symptoms through the examination of their demographic information, isolate etiological agents, and in vitro antifungal susceptibility patterns. Methods: The samples of 170 patients with otitis externa symptoms were collected and confirmed for otomycosis by mycological examination (e.g., potassium hydroxide, methylene blue staining, and fungal culture) and molecular sequencing. In vitro antifungal susceptibility tests against miconazole, fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin were performed according to the Clinical and Laboratory Standards Institute (M27-A3/S4 and M38-A2). Results: Out of 170 patients, 145 subjects (85.29%) showed positive mycological findings. In this study, 55.8% of the patients were male, and the most common age group affected was 50 - 59 years (26.2%). Hearing loss and pruritus were the most common clinical manifestations. The most common occupation was being a housewife (47.5%), and most cases occurred during the winter (40%). Aspergillus niger was the most common species, followed by Aspergillus fumigatus, Candida albicans, and Candida glabrata. Caspofungin showed the highest activity against Aspergillus and Candida isolates; nevertheless, itraconazole demonstrated the lowest activity against Aspergillus isolates. Fluconazole showed the weakest power against Candida species. Conclusions: Due to climatic conditions, humidity, and dust, otomycosis has a high occurrence in Iran. Although otomycosis needs long-term antifungal therapy and recurrence is high in some cases, it is rarely life-threatening, and eardrop antifungals are usually enough to eradicate the infection. Local information about the antifungal pattern is useful for the control, prevention, and treatment of otomycosis.