BackgroundSeveral clinical signs in dermatoscopy are very characteristic of onychomycosis and can be a quick complement for the diagnosis of onychomycosis.ObjectivesThe aim of this study was to evaluate the diagnostic accuracy of dermatoscopy compared to microbiological culture and polymerase chain reaction (PCR), as well as the clinical signs associated with onychomycosis.MethodsThe clinical signs of 125 patients were assessed cross‐sectionally using dermatoscopy, and a positive or negative result was assigned. A sample was then taken for PCR and microbiological culture.ResultsOf the 125 patients, 69.6% (87/125) had positive results when both laboratory tests were combined. When they were not combined, the prevalence was lower at 48% (60/125) with PCR and at 43.2% (54/125) with culture. Furthermore, 76.8% (96/125) were classified as positive with dermatoscopy with a sensitivity of 1, a specificity of 0.76, positive predictive value of 0.91 and negative predictive value of 1 (with 95% confidence intervals). Of the 96 dermatoscopy‐positive samples, 36 were negative with PCR (p < 0.001), 42 were negative with culture (p < 0.001) and nine were negative when both tests were combined (p < 0.001). Clinical signs that were significantly associated with the presence of onychomycosis were subungual hyperkeratosis (dermatoscopy: p = 0.004, odds ratio (OR) = 2.438; PCR + microbiological culture: p = 0.004, OR = 3.221), subungual detritus (p = 0.033, OR = 3.01, only with dermatoscopy) and dermatophytoma (dermatoscopy: p = 0.049, OR = 3.02; PCR + microbiological culture: p = 0.022, OR = 2.40).ConclusionsThe results suggest that dermatoscopy is a good tool for the diagnosis of onychomycosis but should be used as a complementary test or for screening patients to be sampled for laboratory testing. The combination of the three tests can lead to a reduction of false‐positive and false‐negative clinical and laboratory results. This allows for early diagnosis and specific treatment based on test results.