| Summary: | Species of <i>Aspergillus</i> (<i>A.</i>) <i>niger</i> complex and <i>A. flavus</i> complex are predominant molds that are causative agents of otomycoses. The goal of this study was to investigate the clinical presentation, diagnostic procedure, and appearance of relapse in patients with <i>Aspergillus</i>-otomycosis, as well as to determine the biofilm production ability of species isolated in relapse. Thirty patients with laboratory evidenced <i>Aspergillus</i>-otomycosis followed by two check-ups (30 and 60 days after initiation of treatment with antimycotics for local application) were included in the study. For isolation and identification of <i>Aspergillus</i> spp. the standard mycological procedure was applied. Results showed very high sensitivity of microscopy, but 16.7% <i>Aspergillus</i> species required the optimal temperature of 27–28 °C for cultivation. Applied statistical cluster analysis showed a defined specific cluster/group of patients with <i>A. niger</i> complex-otomycosis. Sixty days after diagnosis and treatment initiation, six patients had a relapse, with the same species of <i>Aspergillus</i> genus being the cause. To establish the ability of biofilm production, the modified method described by Pierce and Kvasničková was performed, and all six species isolated in the relapse episode had the ability to produce biofilm. Official criteria and recommendations are needed due to the possibility of misdiagnosis, which leads to the prolongation and complication of the disease.
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