Large-scale use of antimicrobials in agriculture and medicine contributes to antibiotic residues in raw foods, the spread of antimicrobial resistance (AMR) and drug pollution, which seriously threatens human health and imposes significant economic burdens on society, suggesting the need for novel therapeutic options that prevent or control zoonoses. In this study, four probiotics were selected to assess their capability to alleviate pathogen-induced damage. Results showed that a simulated gastrointestinal juice and bile toleratedL. plantarumLac16 with high lactic acid secretion can significantly inhibit the growth of multiple zoonotic pathogens. Lac16 also significantly inhibited the biofilm formation and mRNA expression of virulence traits (genes related to virulence, toxins, flagella biogenesis and motility, antibiotic resistance, biofilm formation and AI-2 quorum sensing) of enterohemorrhagicE. coliO157:H7 (EHEC). Furthermore, Lac16 and Lac26 significantly protectedC. elegansagainst zoonotic pathogen-induced (EHEC,S. typhimurium,C. perfringens) deaths. Moreover, Lac16 significantly promoted epithelial repair and ameliorated lipopolysaccharide (LPS)-induced intestinal epithelial apoptosis and barrier dysfunction by activating the Wnt/β-catenin signaling pathway, and markedly reduced LPS-induced inflammatory responses by inhibiting the TLR4/MyD88 signaling pathway. The present results indicate that Lac16 attenuates enterohemorrhagicE. coliinfection-induced damage by inhibiting key virulence traits ofE. coli, promoting epithelial repair and improving intestinal epithelial barrier function, which may be mediated by the activated Wnt/β-catenin signaling pathway and the inhibited TLR4/MyD88 signaling pathway of the intestinal epithelium.