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Low diversity intestinal dysbiosis has been associated with inflammatory bowel disease, including patients with ulcerative colitis with an ileo-anal pouch anastomosis. Furthermore, specific <i>Escherichia coli</i> phylogroups have been linked to inflammatory bowel disease. Our aim was to characterize the differences among microbiota and <i>E. coli</i> phylogroups in active and inactive pouchitis. Disease activity was assessed using the modified pouch disease activity index and by fecal calprotectin. Microbiota diversity was assessed by 16S rDNA MiSeq sequencing. <i>E. coli</i> phylogroup was determined after triplex PCR. Twenty patients with ulcerative colitis with an ileo-anal pouch anastomosis were included, 10 of whom had active pouchitis. Ileo-anal pouch anastomosis patients had an increased abundance of Proteobacteria colonization compared to patients with ulcerative colitis or Crohn’s disease and healthy controls, <i>p</i> = 1.4·10⁻⁵. No differences in <i>E. coli</i> phylogroup colonization could be determined between cases of active and inactive disease. No significant link was found between α-diversity and pouch inflammation. However, higher levels of Fusobacteria colonization were found in patients with a pouch with a fecal calprotectin level above 500, <i>p</i> = 0.02. In conclusion, patients with a pouch had an increased Proteobacteria abundance, but only Fusobacteria abundance was linked to inflammation.