Fungal otitis externa, an infection of the external auditory canal caused by molds and yeasts, accounts for approximately 10-20% of ear canal infections accompanying high recurrence. The purpose of the current study was to assess the pattern of etiological agents of otomycosis and resistance profile as well as the rate of tympanic membrane perforation. A total of 1040 patients with symptoms of fungal otitis externa, in a period of two years, were investigated. The mycological tests revealed the presence of different fungi in 237 ears (22.8%). Fungal otitis was more related to filamentous fungi of the species Aspergillus flavus (54.43%), A. tubingensis (10.97%), and A. niger (8.86%), followed by yeasts, Candida orthopsilosis (7.59%), C. albicans (6.75%), and C. parapsilosis (5.06%). Tympanic membrane perforation rate was found to be 6.75% and was more common with otomycosis caused by A. flavus, A. tubingensis and C. albicans. In antifungal susceptibility tests, all tested drugs showed generally good activity against most isolates of molds and yeasts, while tolnaftate, clotrimazole, nystatin, and terbinafine had lowest effects. We found that among Aspergillus isolates, one A. niger isolate was resistant to voriconazole, and one A. flavus isolate was resistant to amphotericin B. Furthermore, among Candida species, three isolates of C. orthopsilosis showed high MIC values to fluconazole, two C. albicans isolates were considered fluconazole resistant and one isolate of C. parapsilosis was resistant to caspofungin and 3 isolates were resistant to fluconazole. Regarding the existence of the cases with perforated tympanic membrane and emerging species causing fungal otitis in the current report, the importance of the early physical examination, precise molecular identification, and the antifungal susceptibility evaluation is highlighted.