PurposeDermatophytes are fungi that cause infections affecting hair, nail, and skin; in nails they cause onychomycosis, while in hair they lead to tinea capitis. Detection of dermatophytes using traditional methods, including potassium hydroxide (KOH) and culture on agar-based media leads to high rates of false-negative results. Here, we investigated more accurate diagnostic techniques, including Chicago sky blue staining and Calcofluor white fluorescent staining and compared them with traditional KOH and culture methods for the diagnosis of fungi causing onychomycosis and tinea capitis.Patients and methodsThis study was conducted using samples from 50 patients with dermatophytosis of the hair or nail. Samples were subjected to all the following laboratory investigations: KOH wet mount, culture on Sabouraud’s dextrose agar, Chicago sky blue staining, and Calcofluor white staining. The results of the new diagnostic techniques were compared with those of the traditional methods.ResultsCalcofluor white stain and Chicago sky blue stain for dermatophytosis of hair and nail are more specific and sensitive as compared to traditional diagnostic methods. KOH wet mount is simple, rapid, and inexpensive test but lacks color contrast and gave more false positive (artifacts) and false-negative results as compared to these new stain methods.ConclusionChicago sky blue and Calcofluor white staining are excellent methods for diagnosis of fungal infections, including those that cannot be confirmed using conventional methods.