There is an upsurge of interest in gastro-intestinal microbiology to improve the balance between positive and negative commensals. Mucosal bacteria make closer contact with the host than luminal ones and can therefore have a stronger health impact. An in vitro adhesion assay was developed to study the mucin colonization of bacteria from the mixed microbial communities of the Simulator of the Human Intestinal Microbial Ecosystem. Adhesion capacity differed substantially between bacteria and decreased from lactobacilli over fecal coliforms, bifidobacteria, and clostridia to total anaerobes. Lactobacillus rhamnosus GG adhered most selectively. Further, intestinal water lowered adhesion compared to phosphate-buffered saline. By processing the data to an Adhesion-Related Prebiotic Index, it was found that intestinal water stimulated adherence of positive commensals. Arabinoxylans decreased the adhesion capacity matrix independently, whereas inulin had less or no influence. Measurements of bacterial surface tension, surface hydrophobicity, liquid surface tension, and viscosity showed that bacterial adhesion to mucin agar is a matter of both non-specific and specific interactions. The developed methodology can be useful for the characterization of the relevant but barely investigated mucin-associated bacterial community in health and disease (e.g., IBD) as well as for its modulation with functional foods like prebiotics.