Background and objective Dermatophytosis refers to superficial skin fungal infection in humans. The laboratory diagnosis of the pathogens causing dermatophytosis is challenging. In recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as an effective tool for diagnosing and identifying microorganisms. This study aimed to compare MALDI-TOF MS with conventional methods of identification to assess the existing knowledge on the diagnostic capabilities of MALDI-TOF MS for dermatophyte infections. The findings of this study have significant implications for clinical practice, public health, and future research endeavors in dermatophytosis diagnostics. Methods This prospective study was conducted in the Post Graduate Department of Microbiology at a tertiary-level teaching Medical University over 12 months, from January 1, 2020, to December 31, 2020. A total of 138 clinical cases among the patients attending the skin OPD with clinical features suggesting fungal skin infections were included. Conventional methods such as microscopy, slide culture, biochemical tests, and culture on Sabouraud dextrose agar (SDA) were used as the reference standard for species identification. ATCC (American Type Culture Collection) strains of dermatophytes were used for quality control. The MALDI-TOF Vitek MS (BioMerieux, Craponne, France) system was employed for comparison, and the agreement between the two methods was determined. Results Of the 138 samples, 89 were from males and 49 from females. The majority were taken from the skin (103, 75%) samples followed by nails (30, 21%) and hair (5, 4%). Tinea corporis was the most common clinical type, seen in 81 cases (58.70%), followed by onychomycosis seen in 30 (21.74%) cases, and tinea cruris seen in 16 (11.59%) cases. The most prevalent isolate discovered in tinea corporis was Trichophyton tonsurans (T. tonsurans), followed by Trichophyton mentagrophytes (T. mentagrophytes). Traditional methods and MALDI-TOF MS (Vitek MS) had 100% agreement for T. tonsurans, Trichophyton rubrum (T. rubrum), and Microsporum gypseum (M. gypseum). However, there was disagreement between T. mentagrophytes and Trichophyton interdigitale (T. interdigitale), which had 0.00% agreement. Conclusions Our research showed that no single diagnostic approach would be optimal for every situation that was examined. However, traditional procedures remain the most dependable and efficient methods for mycological diagnostics. However, MALDI-TOF identification results are available much earlier than those of traditional methods.