BACKGROUND Numerous studies have shown that in Crohn's disease (CD), the gut microbiota is of great importance in the induction and maintenance of inflammation in the gastrointestinal tract. Until recently, studies have focused almost exclusively on bacteria in the gut. Lately, more attention has been paid to the role of intestinal fungi. AIM To study the gut mycobiome analysis of pediatric patients with CD (in different stages of disease activity) compared to healthy children. METHODS Fecal samples were collected from patients: With active, newly diagnosed CD (n = 50); active but previously diagnosed and treated CD (n = 16); non-active CD and who were in clinical remission (n = 39) and from healthy volunteers (n = 40). Fungal DNA was isolated from the samples. Next, next generation sequencing (MiSeq, Illumina) was performed. The composition of mycobiota was correlated with clinical and blood parameters. RESULTS Candida spp. were overrepresented in CD patients, while in the control group, the most abundant genus was Saccharomyces. In CD patients, the percentage of Malassezia was almost twice that of the control (P < 0.05). In active CD patients, we documented a higher abundance of Debaryomyces hansenii (D. hansenii) compared to the non-active CD and control (P < 0.05) groups. Moreover, statistically significant changes in the abundance of Mycosphaerella, Rhodotorula, and Microidium were observed. The analyses at the species level and linear discriminant analysis showed that in each group it was possible to distinguish a specific species characteristic of a given patient population. Moreover, we have documented statistically significant correlations between: D. hansenii and patient age (negative); C. zeylanoides and patient age (positive); C. dubliniensis and calprotectin (positive); C. sake and calprotectin (positive); and C. tropicalis and pediatric CD activity index (PCDAI) (positive). CONCLUSION Mycobiome changes in CD patients, and the positive correlation of some species with calprotectin or PCDAI, give strong evidence that fungi may be of key importance in the development of CD.