Background: Acute appendicitis is a surgical emergency in which the appendix is surgically removed to prevent peritonitis due to perforation of the appendix. Depending on age and gender, up to 17% of removed appendices do not show the histopathological changes pathognomonic for acute appendicitis and are called 'pseudo-appendicitis'. Intestinal spirochaetes have been reported in up to 12.3% of these non-inflamed appendices obtained from adults. Although children carry the highest risk for acute appendicitis, not much is known on the prevalence of intestinal spirochaetes in children. The aim of this study was to determine whether there is an association between pseudo-appendicitis and appendiceal spirochaetosis in children. Methods: Archival appendix specimens from paediatric patients (less than 18 years old) were obtained from two Dutch hospitals (acute appendicitis, n = 63; pseudo-appendicitis, n = 55; control appendices, n = 33) and microscopically analysed by H&E staining and spirochaete-specific immunohistochemistry and Brachyspira species specific real-time PCR. Results: Five out of 142 appendices were found to be positive, all in male patients: one in the acute appendicitis group, two in the pseudo-appendicitis group and two in the control group. Conclusion: The results obtained do not provide evidence for a role of Brachyspira species infection in the aetiology of acute appendicitis in children.