Spent culture supernatant (SCS) of the probiotic Lactobacillus rhamnosus GG had been reported to exert antibacterial activity against Salmonella typhimurium. However, the chemical identity of the antimicrobial compound(s) responsible remained unknown. A survey of the antimicrobial compounds produced by L. rhamnosus GG was performed. Lactobacillus rhamnosus GG produced a low-molecular weight, heat-stable, non-proteinaceous bactericidal substance, active at acidic pH against a wide range of bacterial species. SCS of L. rhamnosus GG grown in MRS medium contained five compounds that could meet the above description, if present at the appropriate concentration. Based on different experimental approaches, it could be concluded that under the growth conditions tested, the strong antimicrobial activity of L. rhamnosus GG against Salmonella was mediated by lactic acid.