Salmonella is one of the primary causes of food borne illnesses worldwide. In this study, antibacterial properties of rhubarb against Salmonella were investigated. Initial screening showed that rhubarb root ethanol extract strongly inhibited the growth of Salmonella serotype typhimurium, and the chloroform fraction was found to be the most active fraction. Five major Anthrsaquinone derivatives were identified from the chloroform fraction by UPLC-MS/MS, namely emodin, aloe-emodin, rhein, physcion and chysophanol. Of these five compounds, rhein showed the greatest antibacterial activities against S. typhimurium. Time kill curve assay suggested that rhein killed the bacteria in a relatively fast rate. Further investigations on the mechanisms revealed that rhein significantly altered the integrity of the cell membrane, resulting in the loss of barrier function and leakage of the nucleotide. The morphological changes of S. typhimurium treated with rhein were also observed by scanning electron micrographs.