Samples of two hundred finger‐ and toenails cultured after a 70% alcohol disinfection for 2 min were compared with non‐disinfected samples cultured on a classical way. Alcohol treatment caused a significant decrease in isolation of yeast and moulds. Both organisms were cultivated at 25.5 and 31%, respectively, when not treated with alcohol, whereas the isolation rate dropped to 10 and 17.5%, respectively, after alcohol treatment. Isolation of dermatophytes decreased from 27.5 to 23.5% when alcohol disinfection was used. In conclusion, although alcohol disinfection of nail samples in the laboratory effectively decreases the isolation rate of unwanted contaminants on Sabouraud glucose agar without cycloheximide, it hampers the isolation of dermatophytes and pathogenic yeasts on cycloheximide‐containing media.