The role of fungi as commensals has been neglected for long time and only recently few reports have explored the composition and dynamics of the human gut mycobiota. Commensal fungi are important in human health and disease and changes in commensal fungal populations have been shown to deeply affect pathologies not directly related to fungi, such as Inflammatory Bowel Diseases and Cystic Fibrosis. We studied the fungal gut populations of 111 healthy subjects using a culture-based approach characterizing the isolated fungi for commensalism-related traits. Fungi were detected in 80.2% of subjects leading to the identification of 349 different fungal isolates belonging either to Ascomycetes, Basidiomycetes and Zygomycetes. We found 34 different fungal species, some of which previously isolated solely in environmental samples, phenotypically adapted to be putative commensals of the human gastrointestinal tract. The 39.8% of inspected individuals has been found to carry at least one C. albicans isolate, resulting the most abundant and common yeast species found in ours samples. Analyses of fungal populations’ dynamics suggest that the human gut mycobiota is relatively stable through the lifetime of individuals but significantly differ in a gender-related fashion.