SummaryBackground and objectivesOnychomycosis is common and important to distinguish from other nail diseases. Rapid and accurate diagnosis is necessary for optimal patient treatment and outcome. Non‐invasive diagnostic tools have increasing potential for nail diseases including onychomycosis. This study evaluated line‐field confocal optical coherence tomography (LC‐OCT) as a rapid non‐invasive tool for diagnosing onychomycosis as compared to confocal laser scanning microscopy (CLSM), optical coherence tomography (OCT), and conventional methods.Patients and MethodsIn this prospective study 86 patients with clinically suspected onychomycosis and 14 controls were examined using LC‐OCT, OCT, and CLSM. KOH‐preparation, fungal culture, PCR, and histopathology were used as comparative conventional methods.ResultsLC‐OCT had the highest sensitivity and negative predictive value of all methods used, closely followed by PCR and OCT. Specificity and positive predictive value of LC‐OCT were as high as with CLSM, while OCT scored much lower. The gold standard technique, fungal culture, showed the lowest sensitivity and negative predictive value. Only PCR and culture allowed species differentiation.ConclusionsLC‐OCT enables quick and non‐invasive detection of onychomycosis, with advantages over CLSM and OCT, and similar diagnostic accuracy to PCR but lacking species differentiation. For accurate nail examination, LC‐OCT requires well‐trained and experienced operators.