OBJECTIVES:: Crohnʼs Disease (CD) is a chronic relapsing condition possibly caused by a dysbiotic microbiome. About 30–60% of CD patients have antibodies to Saccharomyces cerevisiae (ASCA), but any association with gut microbiota is unexplored. We hypothesized that ASCA positivity would predict a signature microbial status and clinical phenotype. METHODS:: Ileocolonic mucosal biopsies were obtained from children with CD (n = 135), and controls without inflammatory bowel disease (n = 45). Comparison was made between ASCA status, microbial diversity and clinical characteristics. RESULTS:: ASCA was highly specific but poorly sensitive for the diagnosis of CD. In patients with CD, ASCA positivity was associated with older age (≥10 years), ileocolonic disease and long-term risk of surgery. Microbial alpha and beta diversity were similar in CD patients with or without ASCA, but significantly less when compared to non-IBD controls. Microbial richness was similar across all three groups. Fourteen bacterial species were associated with ASCA positive CD patients and 14 species with ASCA negative patients (p < 0.05). After using a false discovery rate correction Ruminococcus torques and bacterium Yersinia enterolitica 61 remained significantly associated with CD ASCA positivity (p = 0.0178), while Enterobacter cloacae and Faecalibacterium prausnitzii were significantly associated with CD ASCA negativity (p = 0.0178 and 0.0342). CONCLUSION:: ASCA positive and ASCA negative CD patients have significant differences in gut microbiome composition, which could possibly be influencing the phenotype of the disease.