Aims There is interest in using probiotics such as Lactobacillus species to control canine intestinal infections. The bacterial species should be of canine intestinal origin exhibiting host specificity. Bacterial strains were isolated from dog faecal samples and characterized to select specific probiotics as dietary supplements in feed, promoting health status. Methods and Results Bacterial strains have been screened for their probiotic properties including survival to gastric and pancreatic juices and bile salts, resistance to antibiotics and antipathogenicity. Five of 14 isolated Lactobacillus strains were tolerant to gastric stress. They were also resistant to clindamycin and with a significant antimicrobial capacity towards the pathogenic strains tested, variable according to the strain. They were identified by morphological and molecular characterization comparing the 16S rRNA gene sequence with the blast database. Three strains were identified as Lactobacillus reuteri and two as Lactobacillus johnsonii. Sugar fermentation profiles and adhesion to HT29 epithelial cells have been in vitro verified on L. reuteri AI, chosen as probiotic candidate. Resistance to freeze-drying, production and subsequent in vivo administration evaluating strain permanence, were also performed. No loss of vitality has been recorded due to the freeze-drying process. The average value of recovery percentage of L. reuteri AI at the end of the administration period and after 1 week of follow-up was respectively 26 center dot 7 and 17 center dot 4% of the total Lactobacillus sp. Conclusions Among several selected probiotic strains, L. reuteri AI proved to be the best probiotic candidate to use as a supplement for dogs. Significance and Impact of the Study Control of intestinal pathogenic micro-organisms in dogs is a growing concern and the selection of autochthonous probiotic bacterial strains to overcome some of the gut problems associated with the modern domestication of animals is a valuable tool.