Dermatophytes are common pathogens in superficial mycoses that are routinely identified by culture or PCR analysis of freshly collected skin, nail or hair specimens. Although clinical samples are normally processed without delay, practical or research issues may necessitate sample storage until later analysis. However, the influence of extended sample storage on the ability to recover fungi by culture vs. PCR analysis has not been specifically studied. Here, a total of 172 dermatological samples collected from 2013–2015 were examined before and after refrigerated storage at 4°C for 10.2–32.3 (mean 25.6) months. By culture, 35% of the dermatophyte-containing fresh samples remained positive at re-examination. At species level, only 19% of initially Trichophyton rubrum-positive samples yielded a positive result after refrigeration, whereas few samples containing Trichophyton violaceum, Microsporum canis or Microsporum audouinii remained culture-positive. Using PCR, 76% of dermatophyte DNA-positive fresh samples were still positive at re-analysis. Notably, 92% of the samples targeted by the T. rubrum DNA primer remained positive after storage. Hence, PCR analysis is more favourable than cultivation with regard to the detectability of dermatophytes in long-term refrigerated clinical samples.