Data on a mushroom based prebiotic supplementation in patients with Crohn's disease (CD) in western population is scarce. In this pilot trial, we aimed to assess the clinical efficacy and fecal microbial compositional and functional alterations associated with 'Mycodigest,' a commercial prebiotic supplement composed of three mushroom extracts. Patients with mild to moderate CD were recruited to a single center, randomized, double-blind, placebo-controlled pilot induction trial. Clinical efficacy using the Harvey-Bradshaw index and biochemical response using C-reactive protein and fecal calprotectin were assessed at week 8 post-intervention. Fecal samples were assessed by DNA shotgun metagenomic sequencing. A multivariable linear mixed effects model was used to assess alteration in fecal microbiome composition and function pre- and post-'Mycodigest' intervention. Clinical response was higher in the 'Mycodigest' intervention (N = 10) compared to the placebo (N = 6) group (80 vs. 16.7%, respectively, p = 0.035). There were no differences in terms of biochemical response within each group pre- and post-intervention. Post-'Mycodigest' intervention, 25 species were found to be differentially abundant compared to baseline, including increase in short chain fatty acid producing bacteria, such as Parabacteroides distasonis (Beta coefficient 0.92, 95% Confidence interval [CI] 0.36-1.47) and Faecalimonas umbilicata (Beta coefficient 0.57, 95% CI 0.23-0.90). Two microbial pathways related to the metabolism of isoprenoid compounds were increased post-'Mycodigest' intervention. Mushroom based prebiotic supplementation in subjects with CD resulted in clinical improvement which may be related to post-intervention favorable compositional and functional microbial alterations.