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Salmonella enteritidisis an important food-borne pathogen. The use of antibiotics is a serious threat to animal and human health, owing to the existence of resistant strains and drug residues. Lactic acid bacteria, as a new alternative to antibiotics, has attracted much attention. In this study, we investigated the antibacterial potential and underlying mechanism of Lactobacillus rhamnosusSQ511 against S. enteritidisATCC13076. The results revealed that L. rhamnosusSQ511 significantly inhibited S. enteritidisATCC13076 growth or even caused death. Laser confocal microscopic imaging revealed that the cell-free supernatant (CFS) of L. rhamnosusSQ511 elevated the reactive oxygen species level and bacterial membrane depolarization in S. enteritidisATCC13076, leading to cell death. Furthermore, L. rhamnosusSQ511 CFS had severely deleterious effects on S. enteritidisATCC13076, causing membrane destruction and the release of cellular materials. In addition, L. rhamnosusSQ511 CFS significantly reduced the expression of virulence, motility, adhesion, and invasion genes in S. enteritidisATCC13076 (P< 0.05), and considerably inhibited motility and biofilm formation capacity (P< 0.05). Thus, antimicrobial compounds produced by L. rhamnosusSQ511 strongly inhibited S. enteritidisgrowth, mobility, biofilm formation, membrane disruption, and reactive oxygen species generation, and regulated virulence-related gene expressions, presenting promising applications as a probiotic agent.