Background: Since accurate identification of dermatophyte species is essential for epidemiological studies and implementing antifungal treatment, overcoming limitations of conventional diagnostics is a fruitful subject. Objectives and methods: In this study, we investigated real‐time polymerase chain reaction(q‐PCR), matrix‐assisted laser desorption/ionisation time of flight mass spectrometry (MALDI‐TOF MS) and nano‐electrospray ionisation mass spectrometry (nano‐ESI‐MS) to detect and identify the most frequently isolated dermatophytes from human and animal dermatophytosis in comparison with conventional methods. Results: Among 200 samples, the identified species were Microsporum canis (78.22%), Trichophyton verrucosum (10.89%) and T. mentagrophytes (5.94%). Q‐PCR assay displayed great execution attributes for dermatophytes detection and identification. Using MALDI‐TOF MS, M. canis, but none of T. violacium, T. verrucosum or T. mentagrophytes, could be identified. Nano‐ESI‐MS accurately identified all species. The potential virulence attributes of secreted proteases were anticipated and compared between species. Secreted endoproteases belonging to families/subfamilies of metalloproteases, subtilisins and aspartic protease were detected. The analysed exoproteases are aminopeptidases, dipeptidyl peptidases and carboxypeptidases. Microsporum canis have three immunogenic proteins, siderophore iron transporter mirB, protease inhibitors, plasma membrane proteolipid 3 and annexin. Conclusion: In essence, q‐PCR, MALDI‐TOF MS and nano‐ESI‐MS assays are very nearly defeating difficulties of dermatophytes detection and identification, thereby, supplement or supplant conventional diagnosis of dermatophytosis.