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Background: Ulcerative colitis (UC) is a multifactorial chronic inflammatory bowel disease (IBD) that affects large intestine with superficial mucosal inflammation. A dysbiotic gut microbial profile has been associated with UC. Our study aimed to characterize the UC gut bacterial, fungal and metabolic fingerprints by omic approaches. Methods: The 16S rRNA- and ITS2-based metataxonomics and gas chromatography-mass spectrometry/solid phase microextraction (GC–MS/SPME) metabolomic analysis were performed on stool samples of 53 UC patients and 37 healthy subjects (CTRL). Univariate and multivariate approaches were applied to separated and integrated omic data, to define microbiota, mycobiota and metabolic signatures in UC. The interaction between gut bacteria and fungi was investigated by network analysis. Results: In UC cohort we reported the increase of Streptococcus, Bifidobacterium, Enterobacteriaceae, TM7-3, Granulicatella, Peptostreptococcus, Lactobacillus, Veillonella, Enterococcus, Peptoniphilus, Gemellaceae, phenylethyl alcohol, and the decrease of Akkermansia, Ruminococcaceae, Ruminococcus, Gemmiger, Methanobrevibacter, Oscillospira, Coprococus, Christensenellaceae, Clavispora, Vishniacozyma, Quambalaria, hexadecane, cyclopentadecane, 5-Hepten-2-ol, 6 methyl, 3-Carene, caryophillene, p-Cresol, 2-Butenal, indole, 3-methyl-, 6-Methyl-3,5-heptadiene-2-one, 5-Octadecene, and 5-Hepten-2-one, 6 methyl. The integration of the multi-omic data confirmed the presence of a distinctive bacterial, fungal and metabolic fingerprint in UC gut microbiota. Moreover, the network analysis highlighted bacterial and fungal synergistic and/or divergent interkingdom interactions. Conclusions: In this study, we identified intestinal bacterial, fungal and metabolic UC-associated biomarkers. Furthermore, evidence on the relationships between bacterial and fungal ecosystems provides a comprehensive perspective on intestinal dysbiosis and ecological interactions between microorganisms in