Phospholipid-based nanotube-like structures (pLN) were assembled onto the surface of serum albumin nanoparticles (NPs) for potential use as an antimicrobial agent. The resulting pLN-NPs demonstrated dose-dependent antibacterial activity against various pathogens, including Gram-negative and Gram-positive bacteria. The pLN-NPs were found to act via a membrane-destabilizing mechanism, inducing rapid loss of bacterial membrane integrity and subsequent cell death. Furthermore, co-treatment with conventional antibiotics significantly enhanced their bactericidal efficacy. A foodborne pathogen, Vibrio vulnificus, was further examined for its cellular and pathogenic responses to pLN-NPs treatment. The membrane stability of V. vulnificus cells exposed to pLN-NPs significantly decreased, potentially causing membrane disruption. Intraperitoneal injection of pLN-NPs did not induce apparent toxicity in mice, although transient accumulation was observed in several organs. In a V. vulnificus infection mouse model, administration of pLN-NPs significantly reduced mortality in a dose-dependent manner, correlating with decreased numbers of colonizing V. vulnificus. Furthermore, co-therapy of pLN-NPs with the conventional antibiotics in vulnificus-infected mice decreased their mortality in a synergistic manner. Therefore, the pLN-NPs developed in this study offer a promising strategy for controlling antibiotic-resistant bacteria.