Alterations in the intestinal microbial flora have been linked with autoimmune diseases. Our objective was to analyse the composition of the faecal microbiome of children with new-onset juvenile idiopathic arthritis (JIA) compared to healthy controls, and to identify specific gut bacteria associated with JIA. Stool samples from patients were taken at the time of diagnosis of JIA. The microbiome profiles of samples of 30 children with JIA (mean age 6.2 years, 22 girls) were analysed with 16S region-based sequencing profiling and compared to the stool samples of healthy controls (n = 27, mean age 5.4 years, 18 girls). The proportion of bacteria belonging to the phylum Firmicutes was significantly lower in children with JIA [21 % (95 % confident interval [CI]: 17-25 %)] compared to controls [33 % (95 % CI: 26-41 %), p = 0.009]. Bacteria belonging to Bacteroidetes were significantly more abundant in JIA [78 % (95 % CI: 74-82 %)] than in control samples [65 % (95 % CI: 57-73 %), p = 0.008]. Shared operational taxonomic units (OTUs) between the groups revealed that genera Actinobacteria and Fusobacteria were present only in JIA patients and Lentisphaerae only in controls. In summary, faecal flora in JIA is characterised by a low level of Firmicutes and an abundance of Bacteroidetes, resembling the aberration reported in type 1 diabetes. We suggest that alterations in the intestinal microbial flora may challenge the mucosal immune system of genetically susceptible subjects predisposing to local proinflammatory cascades, thus contributing to the development of JIA.