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Microbiota composition may play a role in the development, prognosis, or post-infection of COVID-19. There are studies evaluating the microbiota composition at the time of diagnosis and during the course of COVID-19, especially in adults, while studies in children are limited and no study available in children with multisystem inflammatory syndrome in children (MIS-C). This study was planned to compare intestinal microbiota composition in children diagnosed with MIS-C and acute COVID-19 infection with healthy children. In this prospective multicenter study, 25 children diagnosed with MIS-C, 20 with COVID-19 infection, and 19 healthy children were included. Intestinal microbiota composition was evaluated by 16 s rRNA gene sequencing. We observed changes of diversity, richness, and composition of intestinal microbiota in MIS-C cases compared to COVID-19 cases and in the healthy controls. The Shannon index was higher in the MIS-C group than the healthy controls (p < 0.01). At phylum level, in the MIS-C group, a significantly higher relative abundance of Bacteroidetes and lower abundance of Firmicutes was found compared to the control group. Intestinal microbiota composition changed in MIS-C cases compared to COVID-19 and healthy controls, and Faecalibacterium prausnitzii decreased; Bacteroides uniformis, Bacteroides plebeius, Clostridium ramosum, Eubacterium dolichum, Eggerthella lenta, Bacillus thermoamylovorans, Prevotella tannerae, and Bacteroides coprophilus were dominant in children with MIS-C. At species level, we observed decreased Faecalibacterium prausnitzii, and increased Eubacterium dolichum, Eggerthella lenta, and Bacillus thermoamylovorans in children with MIS-C and increased Bifidobacterium adolescentis and Dorea formicigenerasus in the COVID-19 group. Our study is the first to evaluate the microbiota composition in MIS-C cases. There is a substantial change in the composition of the gut microbiota: (1) reduction of F. prausnitzii in children with MIS-C and COVID-19; (2) an increase of Eggerthella lenta which is related with autoimmunity; and (3) the predominance of E. dolichum is associated with metabolic dysfunctions and obesity in children with MIS-C., Conclusions:  Alterations of the intestinal microbiota might be part of pathogenesis of predisposing factor for MIS-C. It would be beneficial to conduct more extensive studies on the cause-effect relationship of these changes in microbiota composition and their effects on long-term prognosis., What Is Known: • Microbiota composition may play a role in the development, prognosis, or post-infection of COVID-19.  • However, the number of studies on children is limited, and no study on multisystem inflammatory syndrome in children is currently available (MIS-C)., What Is New: • In individuals with MIS-C, the composition of the gut microbiota changed dramatically. • Decreased Faecalibacterium prausnitzii have been observed, increased Eggerthella lenta, which was previously linked to autoimmunity, and predominance of Eubacterium dolichum which was linked to metabolic dysfunction and obesity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)