Background: Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, causes serious systemic disease in humans. Antibiotic treatment is required for the S. Typhi infection, while the inappropriate use of antibiotics causes increased drug-resistant S. Typhi. Hence, alternative therapies through non-antibiotic approaches are urgently needed. The use of beneficial lactic acid bacterium and/or its metabolites to control typhoid fever represent a promising approach, as it may exert protective actions through various mechanisms. Method: In this study, the cell-free culture supernatant (CFCS) of Weissella confusa WM36 was evaluated via the antibacterial activity, and its metabolites were identified. In addition, the effects of CFCS on Salmonella virulence behaviors were also investigated. Result: Based on strong inhibition the growth of S. Typhi DMST 22842, organic acids (lactic acid and acetic acid) and 2,4-Di-tert-butylphenol (2,4 DTBP), were the main antibacterial metabolites presented in CFCS of strain WM36. Minimum inhibitory concentration (MIC) at 40% WM36–CFCS dramatically reduced the S. Typhi population to more than 99.99% at 4 h and completely inhibited biofilm formation, while sub-MIC at 20% (v/v) and MIC could reduce 100% of motility. Additionally, sub-MIC at only 10% (v/v) WM36–CFCS did down-regulate the expression of virulence genes which are responsible for the type-III secretion system, effector proteins, and quorum sensing system in this pathogen. Conclusion: W. confusa WM36 and its metabolites are shown to be a promising candidates, and an effective approach against typhoid Salmonella burden.