Fungal cultures are traditionally incubated for 4 weeks or longer to maximise the recovery of slowly growing fungi. However, the data in support of this are scarce. The objectives of this study were to determine the optimum incubation time for specimens in which moulds or yeast are suspected and to review the literature. A total of 3036 fungal cultures of 2216 dermatological and 820 non-dermatological specimens were analysed. The day on which fungal growth was first noted, was recorded. Eleven of 820 non-dermatological specimens were positive after day 14; in 10 cases, the fungus was considered clinically non-relevant and in one case, the cerebrospinal fluid of a patient receiving therapy for cryptococcosis was positive with Cryptococcus neoformans. Fourteen and three of 2216 dermatological samples showed the growth of a dermatophyte in the third week and fourth week respectively. The results indicate that for specimens sent for the detection of yeast or moulds (except dermatophytes and systemic dimorphic fungi), an incubation period of 2 weeks is sufficient, whereas for dermatophytes, a 4-week incubation period is necessary. Based on these results and previous literature, an algorithm for the incubation time of fungal cultures is proposed.