Background: Appendicitis seems to be a disease of infectious origin, but the detailed pathogenesis is unknown. We aimed to investigate the microbiome of the appendix lumen in patients with and without appendicitis, including a comparison of the subgroups of complicated versus uncomplicated appendicitis. Methods: This prospective observational cohort study included adult patients undergoing laparoscopic appendectomy for suspected appendicitis. According to histopathologic findings, the investigated groups consisted of patients with and without appendicitis, including subgroups of complicated versus uncomplicated appendicitis based on the surgical report. A swab of the appendix lumen was analyzed for genetic material from bacteria with shotgun metagenomics, and outcomes included analyses of microbiome diversity and differential abundance of bacteria. Results: A total of 53 swabs from patients with suspected appendicitis were analyzed: 42 with appendicitis (16 complicated) and 11 without appendicitis. When comparing patients with and without appendicitis, they were equally rich in bacteria (alpha diversity), but the microbiome composition was dissimilar between these groups (beta diversity) (P < .01). No consistent bacterial species were detected in all patients with appendicitis, but a least 3 genera (Blautia, Faecalibacterium, and Fusicatenibacter) and 2 species, Blautia faecis and Blautia wexlerae, were more abundant in patients without appendicitis. For the subgroups complicated versus uncomplicated appendicitis, both measures for microbiome diversity were similar. Conclusion: The appendix microbiome composition of genetic material from bacteria in adult patients with and without appendicitis differed, but the microbiome was similar for patients with complicated versus uncomplicated appendicitis. Trial registration NCT03349814