The gastrointestinal environment is a complex interactive system involving the host, ingested dietary components, and numerous microbial species. We hypothesized that isolation and screening of Lactobacilli and Bifidobacteria adherent to healthy canine gastrointestinal tissue would yield strains with commensal activity in canines. The aims of this study were (1) to isolate a bank of commensal organisms from the canine gastrointestinal tract; (2) to screen these novel microbial isolates for potential probiotic effects; (3) to select one organism from these screens and test its impact on the canine microbiota. Lactic acid bacteria (LAB) were isolated from resected canine gastrointestinal tissue and screened in vitro for putative probiotic activities. Murine studies examined gastrointestinal transit and inhibition of Salmonella typhimurium translocation. One strain was progressed to a canine study where its impact on the gastrointestinal microbiota was determined. Of the 420 isolates from the canine gut, 62 strains were characterised as LAB. Following assessment of the strain bank with regard to pH sensitivity, bile resistance, pathogen inhibition and survival following freeze-drying, four Lactobacillus strains and two Bifidobacteria strains were selected for further examination. Bifidobacterium animalis AHC7 adhered to epithelial cells, transited the murine gastrointestinal tract to high numbers and significantly reduced S. typhimurium translocation. B. animalis AHC7 consumption significantly reduced the carriage of Clostridia, in particular Clostridium difficile, in dogs. This study describes the isolation and screening of canine-derived bacterial strains with commensal traits. The results demonstrate that B. animalis AHC7 has significant potential for improving canine gastrointestinal health.