Background: Bacteria are implicated as important factors in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to seek evidence of possible bacterial targets of the immune response related to IBD in children. Methods: Seventy-eight children referred to the Department of Paediatrics at Tampere University Hospital on suspicion of IBD were included in the study. Upper and lower gastrointestinal endoscopies with biopsies were performed on all children. Sera from 75 children were tested for antibodies to the Pseudomonas fluorescens-associated sequence I2, a Bacteroides caccae TonB-linked outer membrane protein, OmpW, anti-Saccharomyces cerevisiae, and perinuclear anti-neutrophil cytoplasmic antibodies. Results: The IBD diagnosis was confirmed in 35 children (18 with Crohn's disease [CD], 12 with ulcerative colitis [UC], and 5 with indeterminate colitis [IC]); 43 children were found to have no inflammation in the gut. Forty-three percent (15 of 35) of those with IBD evinced positive seroreactivity to I2 and 46% (16 of 35) to OmpW. In CD, seroreactivity to I2 and OmpW was 50% (9 of 18) and 61% (11 of 18), respectively. Serum anti-I2 and anti-OmpW immunoglobulin A levels were significantly elevated in children with CD in comparison with the non-IBD group (P = 0.007 and P = 0.001, respectively). A combination of OmpW, I2, and anti-S cerevisiae tests identified 94% of CD patients, and a combination of OmpW, I2, and perinuclear anti-neutrophil cytoplasmic antibodies detected 83% of UC cases. Conclusions: Among children with IBD, strong serological responses to microbial antigens can be found, suggesting that P fluorescens and B caccae antigens have a potential role in the microbiology and immunology of the disease. Furthermore, serologic reactivity to the set of antigens studied here seems to be applicable in the initial differential diagnosis of children with CD and UC.