This study investigated the antimicrobial efficacy of a cell-free supernatant (CFS) and live cells from a mixture of Limosilactobacillus fermentum strains (139, 296, and 263). These strains are candidates for probiotic use and were evaluated against five STb virotype enterotoxigenic Escherichia coli (ETEC) strains. Antagonistic activities of L. fermentum mixed cells and CFS were assessed with the spot agar method. The impacts of CFS on viable cell counts in laboratory medium, biofilm formation, cell surface hydrophobicity, mucin, and motility were evaluated. The effects of CFS on ETEC viable cell counts under laboratory medium and during exposure to simulated gastrointestinal conditions were measured using plate count, and the physiological status of L. fermentum cells was assessed with flow cytometry. The nuclear magnetic resonance technique identified the metabolites from CFS and its effects on the ETEC metabolite profile. A larger growth inhibition zone was caused by L. fermentum viable cell mixture (18.4-35.8 mm) than CFS (10.1-30.1 mm). CFS caused viable cell count reductions of ≥ 5 log CFU/ml at 24 h of exposure and reduced biofilm formation, cell surface hydrophobicity, mucin, and swimming and swarming motility of ETEC strains. The exposure of ETEC strains to simulated gastrointestinal conditions resulted in viable cell counts <
2 log CFU/ml at the final digestion when supplemented with CFS. Exposure to CFS resulted in sizes of ETEC cell subpopulations characterized as dead or injured >
80.0%. Lactate and acetate were the most critical variables in CFS, directing decreased ETEC metabolic activity. Results showed that the examined CFS from L. fermentum 139, 263, and 296 mixture was an effective antimicrobial against ETEC.