The present study was designed to identify the microbial community as well as to analyze its diversity by means of metagenomic Next Generation Sequencing (mNGS) in 17 patients with otomycosis treated with terbinafine in the Department of Otolaryngology of Shandong Provincial Hospital from June 2021 to June 2022, so as to evaluate the relationship between microbial community and terbinafine resistance. Those 17 patients were divided into two groups, i.e., Terbinafine Effective Group (TEG, n = 14 cases) and Terbinafine Resistance Group (TRG, n = 3 cases) according to the therapy effect, whose microbial community of secretion of external auditory canal was identified using mNGS. We found that the sequence of bacteria was significantly more than that of fungi and, whereas, the difference between the two groups of bacteria was not significant. There were significant differences in fungal community between the two groups. Aspergillus was the main pathogenic fungus of TEG patients while Malassezia was a dominant fungus in TRG patients. In conclusion, the results from this work indicate that Aspergillus terreusis is the main pathogenic fungus in this cohort of otomycosis patients and MNGS sequencing can offer comprehensive information about the microbial community of otomycosis. The fungus community dominated by Malassezia is more likely to be resistant to terbinafine, which provides certain guidance for clinical treatment of otomycosis with terbinafine.