Certain strains of Lactobacillus, which have therapeutic and nutritional value, may facilitate the reduction of bacterial translocation in hosts under critical conditions. The effects may vary between different strains of Lactobacillus. The aim of this study was to evaluate and compare the impact of three Lactobacillus strains on methotrexate-induced enterocolitis in rats. From the beginning of the study, rats received an intragastric infusion of an elemental diet alone or supplemented with Lactobacillus plantarum DSM 6595, Lactobacillus rhamnosus DSM 6594, or Lactobacillus acidophilus DSM 20079 in fermented oatbase. Methotrexate (20 mg/kg) was injected intraperitoneally to all animals on the 4th day, and sampling was done on the 7th day. Supplementation of all three Lactobacillus strains significantly lowered the bacterial translocation to mesenteric lymph nodes, liver and blood, which was accompanied by a reduction in overgrown pathogenic bacteria and an increase in lactobacilli in rats with enterocolitis. L. plantarum and L. rhamnosus were more effective in reducing the bacterial translocation and improving the gut mucosal protein, DNA and RNA content than L. acidophilus. The present results may imply that L. plantarum and L. rhamnosus have more significance than L. acidophilus in future bacteriotherapy.