Onychomycosis is a prevalent disease of the nail. Traditional methods for diagnosis include direct microscopy with potassium hydroxide (KOH microscopy) and fungal culture. Other techniques using histochemical staining have higher sensitivity, but cannot identify genus or species of the infecting agent. PCR assays are sensitive, specific, and capable of genus and species level identification. We describe a real-time PCR assay for 15 different fungi that are associated with onychomycosis. Of 425 clinical samples suspected of onychomycosis analyzed by fungal culture and PCR, 219 samples were positive for both (52% agreement). Of the 206 discordant samples, 95% were resolved in favor of PCR by DNA sequencing. On a larger data set of 2,452 samples, positivity rates for histopathology, PCR, and culture were 85%, 73%, and 54% respectively. Further, 48% of PCR positive and 51% of histopathology positive samples were negative by culture. PCR outperformed culture compared to histopathology for sensitivity (80% versus 49%), specificity (92% versus 79%), positive predictive value (94% versus 77%), and negative predictive value (76% versus 52%). These results indicate the culture method lacks the sensitivity to be a reliable assay for onychomycosis, that PCR and histopathology are highly concordant, and that PCR provides the highest degree of diagnostic accuracy available.