A dermatophyte antigen kit (DQT) was released in Japan as an in vitro diagnostic tool to identify tinea unguium in June 2022. From July 2022 to February 2023, we examined 75 potassium hydroxide (KOH)-negative patients (male, n = 23; female, n = 52; mean ± SD age, 63.6 ± 13.9 years) and determined the accuracy in confirming the fungal element with ZoomBlue™ staining at 400× magnification. The DQT results were classified into three categories. DQT-positive onychomycosis was detected in 27 patients with tinea unguium and two with non-dermatophyte onychomycosis. Fungal cultures were positive in 14 (51.8%) patients (Trichophyton rubrum [n = 11], T. interdigitale [n = 1], Fusarium solani [n = 1], and Talaromyces muroii [n = 1]). DQT-negative onychomycosis included ten patients with cured tinea unguium and 3 with Candida onychomycosis. Twenty-three patients had DQT-negative mimics for onychomycosis (onychauxis [n = 11], traumatic onycholysis [n = 8], yellow nail syndrome [n = 5], pincer nail deformity [n = 3], brittle nail syndrome [n = 2], contact dermatitis [n = 2], lichen planus [n = 1] and psoriasis [n = 1]). Because sparse, atrophic and/or fragmented mycelia are invisible in direct microscopy with potassium hydroxide (KOH) at 100× magnification, DQT was beneficial for diagnosing onychomycosis.