Otomycosis is worldwide in distribution and most commonly caused by Aspergillus species. Amphotericin B, itraconazole and voriconazole are used for the treatment of aspergillosis, but recently an increase in resistance to these agents has been reported. We aimed at investigating the in vitro activities of amphotericin B, voriconazole and itraconazole against Aspergillus isolates causing otomycosis. Mycological analysis of samples from the ear canals of patients was performed by culturing onto Sabouraud Dextrose Agar and by evaluating microscopically. Aspergillus species were identified with colony morphology and microscopic appearance, and tested for susceptibilities to amphotericin B, itraconazole and voriconazole by the CLSI reference broth microdilution method (M38-A document). A total of 120 isolates from 120 patients, comprising 57 Aspergillus niger, 42 Aspergillus fumigatus, nine Aspergillus flavus, six Aspergillus nidulans and six Aspergillus terreus strains were tested. No resistance was determined against amphotericin B and voriconazole, while six A. fumigatus and three A. niger isolates were resistant to itraconazole. In vitro data obtained in this study showed the resistance to itraconazole, while all of the isolates were susceptible to voriconazole and amphotericin B. Voriconazole seemed to be an alternative in the treatment of infections related to Aspergillus spp. but further studies are needed to learn more about the antifungal resistance of different species of Aspergillus to different agents.