Studies so far conducted on irritable bowel syndrome (IBS) have been focused mainly on the role of gut bacterial dysbiosis in modulating the intestinal permeability, inflammation, and motility, with consequences on the quality of life. Limited evidences showed a potential involvement of gut fungal communities. Here, the gut bacterial and fungal microbiota of a cohort of IBS patients have been characterized and compared with that of healthy subjects (HS). The IBS microbial community structure differed significantly compared to HS. In particular, we observed an enrichment of bacterial taxa involved in gut inflammation, such as Enterobacteriaceae, Streptococcus, Fusobacteria, Gemella, and Rothia, as well as depletion of health-promoting bacterial genera, such as Roseburia and Faecalibacterium. Gut microbial profiles in IBS patients differed also in accordance with constipation. Sequence analysis of the gut mycobiota showed enrichment of Saccharomycetes in IBS. Culturomics analysis of fungal isolates from feces showed enrichment of Candida spp. displaying from IBS a clonal expansion and a distinct genotypic profiles and different phenotypical features when compared to HS of Candida albicans isolates. Alongside the well-characterized gut bacterial dysbiosis in IBS, this study shed light on a yet poorly explored fungal component of the intestinal ecosystem, the gut mycobiota. Our results showed a differential fungal community in IBS compared to HS, suggesting potential for new insights on the involvement of the gut mycobiota in IBS.