Intestinal microbiota is involved in metabolic processes and the pathophysiology of various gastrointestinal disorders. We aimed to characterize the microbiome of the appendix in acute pediatric appendicitis comparing extraluminal and intraluminal samples.Between January and June 2015, 29 children (3-17 years, mean age 10.7 ± 3.4 years, sex M:F = 2.6:1) undergoing laparoscopic appendectomy for acute appendicitis were prospectively included in the study. Samples for bacterial cultures (n = 29) and 16S ribosomal desoxyribonucleic acid (rDNA) sequencing (randomly chosen n = 16/29) were taken intracorporeally from the appendiceal surface before preparation ("extraluminal") and from the appendiceal lumen after removal ("intraluminal"). The degree of inflammation was histologically classified into catarrhal, phlegmonous, and gangrenous appendicitis.Seventeen bacterial species were cultivated in 28 of 29 intraluminal samples and 4 species were cultivated in 2 of 29 extraluminal samples. Using 16S rDNA sequencing, 267 species were detected in intraluminal but none in extraluminal samples. Abundance and diversity of detected species differed significantly between histological groups of acute appendicitis in bacterial cultures (P = .001), but not after 16S rDNA sequencing.The appendiceal microbiome showed a high diversity in acute pediatric appendicitis. The intraluminal microbial composition differed significantly depending on the degree of inflammation. As bacteria were rarely found extraluminally by culture and not at all by sequencing, the inflammation in acute appendicitis may start inside the appendix and spread transmurally.