| Background: Dermatophytosis, a prevalent fungal infection in urban settings, poses diagnostic and therapeutic challenges due to its high incidence and rising antifungal resistance. Accurate identification of dermatophyte species is crucial for effective management, particularly in densely populated areas like Silvassa, India. This study combines conventional and molecular methods to isolate and identify dermatophytes from clinical skin samples in an urban population. Objective: To determine the prevalence and species distribution of dermatophytes in urban patients at NAMO Hospital, Silvassa, and to evaluate the efficacy of molecular identification using ITS region sequencing compared to conventional culture techniques. Methods: Between January and June 2025, 150 skin scrapings were collected from patients with suspected dermatophytosis at NAMO Hospital. Samples underwent direct microscopy with 10% KOH, followed by culture on Sabouraud’s dextrose agar supplemented with chloramphenicol and cycloheximide. Isolates were identified morphologically and confirmed by PCR amplification and sequencing of the ITS region. Prevalence and species distribution were analyzed, with statistical significance assessed using chi-square tests (p < 0.05). Results: Of the 150 samples, 92 (61.3%) were culture-positive for dermatophytes. Trichophyton rubrum was the most common species (42 isolates, 45.7%), followed by Trichophyton mentagrophytes (26, 28.3%), Microsporum canis (13, 14.1%), Epidermophyton floccosum (8, 8.7%), and Trichophyton tonsurans (3, 3.3%). Males (65.2%) and the 25–40 age group (52.2%) showed higher prevalence (p = 0.03). Molecular sequencing resolved identification ambiguities in 14 isolates (15.2%) and detected mixed infections in 10 cases (10.9%). A slight increase in cases was noted during monsoon months. Conclusion: The high prevalence of dermatophytosis in urban Silvassa, dominated by T. rubrum, underscores the need for precise diagnostics. Molecular identification via ITS sequencing enhances accuracy and speed, offering a valuable tool for epidemiological surveillance and clinical management in urban settings. These findings advocate for integrating molecular methods into routine diagnostics to address the growing challenge of dermatophytosis in India.