AIMS: This study assessed the antibacterial, anti-biofilm, and immunomodulatory activity of Lactobacillus acidophilus (strain Scav) postbiotic (LaP) in a mouse model of Pseudomonas aeruginosa wound infection and evaluated the bioactive components of the LaP. METHODS AND RESULTS: LaP was tested for P. aeruginosa clearance and immunomodulatory activity during wound infection. We show that LaP applied 1 hour after infection reduced tissue bacterial burden within 24 hours, and this reduction persisted for 5 days. Ciprofloxacin given once at the exact same time did not reduce bacteria load as compared to vehicle controls. LaP reduced plasma IL-6 and MCP-1 levels after 5 days. Wound tissue IL-6 and MCP-1 levels were increased in infected vehicle mice at 5 days, but tissues from LaP treated mice were similar to sham controls. LaP increased tissue IL-10 (anti-inflammatory cytokine) levels. Ciprofloxacin decreased plasma and tissue IL-6 compared to vehicle controls but did not affect MCP-1 or IL-10 levels. To elucidate antibacterial and anti-biofilm metabolite(s) in LaP, fractionation followed by P. aeruginosa antagonistic activity assays were performed. This was followed by liquid chromatography coupled to mass spectrometry (LCMS) analysis. Our analyses identified a low molecular weight, polar molecule which had both antibacterial and anti-biofilm activity. CONCLUSIONS: L. acidophilus secretes an antibacterial and anti-biofilm metabolite that reduced pathogen burden, resolved systemic inflammation in a P. aeruginosa wound infection model.