Onychomycosis is a recalcitrant fungal infection of the nail unit that can lead to secondary infections and foot complications. Accurate pathogen identification by confirmatory testing is recommended to improve treatment outcomes. In this study, we reviewed the records of 710,541 patients whose nail specimens were sent to a single molecular diagnostic laboratory between 2015 and 2024. PCR testing revealed a more comprehensive spectrum of pathogens than previously reported, which was corroborated by the demonstration of fungal invasion on histopathology. Consistent with our current understanding, the T. rubrum complex (54.3%) are among the most common pathogens; however, a significant portion of mycology-confirmed diagnoses were caused by the T. mentagrophytes complex (6.5%), Aspergillus (7.0%) and Fusarium (4.5%). Females were significantly more likely to be infected with non-dermatophytes molds (NDMs; OR: 2.0), including Aspergillus (OR: 3.3) and Fusarium (OR: 2.0), and yeasts (OR: 1.5), including Candida albicans (OR: 2.0) and C. parapsilosis (OR 1.6), than males. The T. mentagrophytes complex became more prevalent with age, and conversely the T. rubrum complex became less prevalent with age. Patients aged ≥65 years also demonstrated a higher likelihood of contracting onychomycosis caused by NDMs (OR: 1.6), including Aspergillus (OR: 2.2), Acremonium (OR: 3.5), Scopulariopsis (OR: 2.9), Neoscytalidium (OR: 3.8), and yeasts (OR: 1.8), including C. albicans (OR: 1.9) and C. parapsilosis (OR: 1.7), than young adults. NDMs (e.g., Aspergillus and Fusarium ) and yeasts were, overall, more likely to cause superficial onychomycosis and less likely to cause dystrophic onychomycosis than dermatophytes. With regards to subungual onychomycosis, Aspergillus , Scopulariopsis and Neoscytalidium had a similar likelihood as dermatophytes. The advent of molecular diagnostics enabling a timely and accurate pathogen identification can better inform healthcare providers of appropriate treatment selections and develop evidence-based recommendations.