Currently, the issue of the relationship between gut microbiota and juvenile idiopathic arthritis (JIA) is still relevant. The study was aimed to assess alterations in the gut microbiota taxonomic composition and estimate the relationship between these alterations and cortisol, melatonin, and TNFα at the genus level in patients with JIA. The comparative cross-sectional study involved 65 patients with JIA (index group) and 60 healthy children (control group). The gut microbiota taxonomic composition and plasma levels of cortisol, melatonin, and TNFα were assessed. The following alterations of the gut microbiota taxonomic composition were found in patients with JIA: the significantly decreased abundance of Anaerostipes (р = 0.042), Lachnospira (р = 0.034), Roseburia (р = 0.002), Coprococcus (р = 0.014), Dialister (р = 0.003) and the increase in the abundance of Ruminococcus (р = 0.012). There were significant correlations of cortisol levels with the abundance of Lachnospira (r = –0.44; p = 0.001), melatonin concentrations and the abundance of Coprococcus (r = –0.48; p = 0.023), the levels of TNFα and the abundance of Ruminococcus (r = 0.52; p = 0.001). The association of the Lachnospira, Roseburia, and Ruminococcus abundance with the higher DAS28 scores was discovered (r = –0.57; p = 0.002; r = –0.44; p = 0.002; r = 0.54; p = 0.032, respectively). The findings provide additional information about the features of gut microbiota alterations and their correlation with some hormone and inflammatory biomarkers associated with JIA, that could provide the basis for further research and possibly for new approaches to treatment of this disorder.