Lipoteichoic acid (LTA), a bacterial cell surface macromolecule, plays a key role in probiotic–host interactions by engaging host pattern recognition receptors in the gastrointestinal mucosa. This study investigates the potential of Lactiplantibacillus plantarum LTA in combating intestinal pathogens and supporting gut health. We demonstrate that L. plantarum LTA, acting as a biosurfactant, significantly inhibits biofilm formation by various intestinal pathogens, including Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes. This is achieved by disrupting biofilm structures, reducing extracellular polymer secretion, and downregulating biofilm-related gene expression. Additionally, LTA effectively prevents pathogen adhesion to Caco-2 cells and reduces Escherichia coli-induced apoptosis. LTA also modulates macrophage inflammation by regulating cytokine secretion and decreasing NO and ROS production through the TLR4-MAPK-NF-κB signaling pathway. These findings provide valuable insights into the mechanisms by which L. plantarum LTA supports gut health and highlight its potential applications in preventing intestinal pathogen colonization and enhancing immune modulation.