Background Intestinal barriers, intestinal flora, and mucosal immunity are the main factors responsible for the development of various allergic and autoimmune diseases. The aim of this study was to investigate the relationship between the intestinal flora of children and the presence of type 1 diabetes, and to determine if gut microbiota could partly explain the etiology of the disease. Methods Fecal flora analysis was done using quantitative cultures on selective and non-selective media with different thermal and atmospheric conditions for bacterial and fungal growth. The study group consisted of 35 patients (16 female, 19 male; mean age, 10.73 +/- 4.16 years), who had been followed by the University of Istanbul, Cerrahpasa Medical Faculty, Department of Pediatrics, and were newly diagnosed with type 1 diabetes. The control group consisted of 35 healthy subjects (15 female, 20 male; mean age, 9.96 +/- 4.09 years), who were randomly selected and had similar demographics. Results Bifidobacterium colonization was lower in patients with type 1 diabetes compared to the control group, whereas Candida albicans and Enterobacteriaceae other than Echerichia coli colonization was increased. Conclusion A decrease in beneficial anaerobic bacteria levels and a concomitant increase in Enterobacteriaceae other than E.coli and C.albicans colonization may lead to a disturbance in the ecological balance of intestinal flora, which could be a triggering factor in type 1 diabetes etiology.