Growing evidence supports the efficacy of antimicrobial peptides against foodborne pathogens, though their antimicrobial spectrum and mechanism can vary depending on their origin. We investigated the antimicrobial spectrum of antimicrobial peptides derived from Lactobacillus paracasei A1, their effects on the survival rate and bactericidal mechanisms against Vibrio parahaemolyticus, and identified the functional short peptides within them. The crude extracts of antimicrobial peptides exhibited antibacterial properties against 13 pathogenic bacteria, showing strong inhibition of V. parahaemolyticus by disrupting the structural integrity of cell membranes. At minimum inhibitory concentrations, these peptides significantly disrupted the initial adhesion, membrane formation, and existing biofilms of V. parahaemolyticus, effectively inhibiting pathogen spread, enhancing the efficacy of antimicrobial agents, and reducing food safety risks. LC–MS/MS identification revealed four effective short peptides, all demonstrating potent bacteriostatic effects against V. parahaemolyticus. Our findings indicate that antimicrobial peptides can effectively destroy bacterial structures as well as the stability and regeneration of biofilms, making them promising candidates for use as food additives to control foodborne pathogens.