Background In the absence of a real gold standard, comparative studies are still done on diagnostic methods for onychomycosis. There are only a few attempts using latent class analysis to determine the value of polymerase chain reaction in comparison to conventional methods. We aimed to determine the value of histological examination in such a way for the diagnosis of onychomycosis. Methods Potassium hydroxide mount (KOH), culture and histological examination with periodic acid-Schiff (PAS), and Gomori's methenamine silver (GMS) stains were done in 106 patients having clinically suspected toenail onychomycosis. Results KOH was positive in 74% of the patients; culture in 14%; PAS in 30%; and GMS in 66%. According to the results of the latent class analysis, culture and PAS were highly specific but poorly sensitive; KOH, highly sensitive but poorly specific; and GMS, both highly sensitive and specific. Conclusions Based on these results, we have proposed KOH as a screening test and GMS as a confirmatory test for the diagnosis of onychomycosis in our own practice. However, since positivity rates of different diagnostic methods vary widely in different centers, it is more suitable that every center should determine their own diagnostic strategy by evaluating their own results with latent class analysis.