Introduction: Fungi causing otomycosis are mostly saprophytic in nature. Pathogenicity of fungi is attributable to presence of various virulence factors like biofilm formation, presence of enzymes like urease, phospholipase, hemolysin and production of mycotoxins like aflatoxin. Aim: To compare the toxigenicity, molecular and conventional methods in diagnosis of otomycosis. Methods: All clinically diagnosed and untreated cases of otomycosis were included in the study. History of otalgia, aural pruritus, ear discharge, diminished hearing, ear block, and tinnitus was elicited. Results: Biofilm production was seen with 6 isolates of Candida albicans (29%). Hemolysin was produced in 12 isolates of Aspergillus niger (23.53%). Aspergillus niger species showed higher production of Aflatoxins B1 and G1. 2 of the rare isolates were subjected to PCR followed by Sanger sequencing for speciation and identified as Cladosporidium cladosporioides. Conclusion: PCR and Gene sequencing are reliable methods for identification and speciation of rare fungi. Analysis of virulence factors showed biofilm formation in Candida. Production of Urease and Hemolysin were identified as virulence factors among Aspergillus species.
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