Aims: The anti-infectious activity of lactobacilli against multi-drug resistant Salmonella enterica serovar Typhimurium DT104 (DT104) was examined in a murine model of an opportunistic antibiotic-induced infection. Methods and Results: Explosive intestinal growth and subsequent lethal extra-intestinal translocation after oral infection with DT104 during fosfomycin (FOM) administration was significantly inhibited by continuous oral administration of Lactobacillus casei strain Shirota (LcS), which is naturally resistant to FOM, at a dose of 108 colony-forming units per mouse daily to mice. Comparison of the anti-Salmonella activity of several Lactobacillus type strains with natural resistance to FOM revealed that Lactobacillus brevis ATCC 14869T, Lactobacillus plantarum ATCC 14917T, Lactobacillus reuteri JCM 1112T, Lactobacillus rhamnosus ATCC 7469T and Lactobacillus salivarius ATCC 11741T conferred no activity even when they obtained the high population levels almost similar to those of the effective strains such as LcS, Lact. casei ATCC 334T and Lactobacillus zeae ATCC 15820T. The increase in concentration of organic acids and maintenance of the lower pH in the intestine because of Lactobacillus colonization were correlated with the anti-infectious activity. Moreover, heat-killed LcS was not protective against the infection, suggesting that the metabolic activity of lactobacilli is important for the anti-infectious activity. Conclusion: These results suggest that certain lactobacilli in combination with antibiotics may be useful for prophylaxis against opportunistic intestinal infections by multi-drug resistant pathogens, such as DT104. Significance and Impact of the Study: Antibiotics such as FOM disrupt the metabolic activity of the intestinal microbiota that produce organic acids, and that only probiotic strains that are metabolically active in vivo should be selected to prevent intestinal infection when used clinically in combination with certain antibiotics.