Emerging evidence suggests that high fat diet (HFD) is associated with gut microbiome dysbiosis and related disorders. Bilberry is a prebiotic food component with known health benefits. Herein the dynamics of colonic mucus layer and microbiome during HFD and bilberries supplementation are addressed.The effects on colonic mucus thickness in vivo and gut microbiota composition (Illumina sequencing, quantitative Real-Time PCR) were investigated in young rats fed low or high fat diets with or without bilberries for 8 weeks (n = 8). HFD induced significant local colonic effects, despite no observed weight gain or systemic inflammation, as HFD caused epithelial upregulation of inducible nitric oxide synthase (iNOS), which was counteracted by bilberry. The firmly adherent mucus layer became thicker and the mRNA levels of Muc2 and Tff3 were increased by HFD with or without bilberry. In parallel, HFD reduced the colonic abundance of mucolytic bacterial species Akkermansia muciniphila and Bacteroides spp. Finally, bilberry prevented the HFD-induced microbiota dysbiosis including expansion of pathobionts, e.g., Enterobacteriaceae.High fat diets expand firmly adherent mucus thickness and reduce mucus foraging bacteria populations in colon prior to obesity. Enriching HFD with bilberry protects against intestinal inflammation and marked microbiota encroachment. This article is protected by copyright. All rights reserved.