Abstract Background Adherent-invasive Escherichia coli (AIEC) have been implicated in the ethiopathogenesis of Crohn’s disease (CD). In this study, we analyzed a collection of intestinal mucosa-associated E. coli isolates, presenting AIEC phenotypes, isolated from biopsies of CD pediatric patients and non-inflammatory bowel diseases (IBD) controls, in order to investigate their genetic and phenotypic pathogenic features. Results A total of 616 E. coli isolates from biopsies of four pediatric CD patients and of four non-IBD controls were collected and individually analyzed. For AIEC identification, adherent isolates were assayed for invasiveness, and the capacity of the adhesive-invasive isolates to survive and replicate intracellularly was determined over macrophages J774. In this way we identified 36 AIEC-like isolates. Interestingly, their relative abundance was significantly higher in CD patients (10%; 31/308) than in non-IBD controls (1%; 5/308) ( χ 2 = 38.96 p