Adhesion to the intestinal mucosa is considered to be one of the main selection criteria of lactic acid bacteria for probiotic use. Adhesive probiotics are, for example, considered to provide better antagonism against pathogenic bacteria when compared to non-adhesive strains. Here a new model is described for studying adhesion and interaction of probiotic and pathogenic bacteria in the intestinal mucus in which the intestinal microbiota is present. The model is based on the use of human intestinal tissue, fluorescent-tagged bacteria and confocal laser scanning microscopy (CLSM) in adhesion measurements as well as human intestinal mucus and bioluminescent-tagged bacteria in viability measurements. Use of CLSM enabled, for the first time, real-time three-dimensional observations of live probiotic bacteria in their natural environment, the intestinal mucosa. When the real-time measurement of bacterial adhesion was combined with the real-time sensitive measurement of bacterial viability, it could be studied whether or not the adherent pathogens were alive. The model was used to study the interaction between Lactobacillus rhamnosus GG and Salmonella enterica serovar Typhimurium. We show that L. rhamnosus GG did not affect the adhesion or the viability of S. enterica serovar Typhimurium. Instead S. enterica serovar Typhimurium was shown to decrease the adhesion of L. rhamnosus GG in displacement assays. Moreover, the method is suitable for studies in which the interaction of two or more bacteria is examined in an environment in which other bacteria are present.