Salmonella is a serious foodborne pathogen responsible for more than 90 million cases of gastroenteritis worldwide annually. Due to the gradual increase in antibiotic-resistant Salmonella strains, the identification of natural antibacterial substances is urgently needed. Herein, we purified natural l-phenyl lactic acid (L-PLA) from Lactobacillus plantarum ZJ316 and revealed its antimicrobial mode against Salmonella enterica subsp. enterica ATCC 14028. L-PLA (98.14% pure) was obtained using the macroporous resin XAD-16, solid-phase extraction (SPE), reverse-phase high-performance liquid chromatography (RP-HPLC), and chiral chromatography. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results showed that the cell morphology was severely compromised. Transmembrane electrical potential (Delta psi), transmembrane pH gradient (Delta pH), intracellular ATP level, extracellular electrical conductivity (EC), and genomic DNA analyses were employed to evaluate the antibacterial mode of action of L-PLA. The proton motive force (PMF) and ATP of Salmonella cells rapidly dissipated, and the EC markedly increased. The gel retardation assay demonstrated that L-PLA could bind to genomic DNA and intercalate into the nucleic acids. The anti-Salmonella mode of action of L-PLA was attributed to the destruction of the cell membrane and genomic DNA binding. This research suggests that L-PLA has potential applications as an antimicrobial agent in food, medicine, and other fields.