This study screened and identified a non-cytotoxic Lactobacillus strain, CYLB565, from food samples. In the wound healing assay, this strain enhanced gap closure in GES-1 cells, while its cell-free supernatant promoted gap closure in both GES-1 and HaCaT cells. CYLB565 and its secretions substantially inhibited pathogens at pH 4 and 6.5. In the anti-inflammatory assay, EPS pre-treatment showed preventive potential against inflammation induced by E. coli, LPS, and H. pylori; EPS co-treatment showed immediate inhibitory potential against inflammation induced by E. coli and LPS; and EPS post-treatment showed therapeutic potential against inflammation induced by E. coli and H. pylori. In addition, CYLB565 has gastrointestinal tolerance at pH 3 and in medium with 0.3% bile salts. According to whole genome sequence results, CYLB565 is a newly identified Lactiplantibacillus plantarum strain that has not been previously reported in public strain databases. Its plasmids are genetically stable and considered safe for probiotic use. CYLB565 may be suitable for preventing and treating bacterial gastroenteritis. This study highlights that cooperative host-microbiota interactions and competitive dynamics among commensal and pathogenic bacteria may represent novel strategies for treating wounds and inflammation.