Vibrio vulnificus causes fatal infections in humans, and antibiotics are commonly used in treatment regimens against V. vulnificus infection. However, the therapeutic effects of antibiotics are limited by multidrug resistance. In this study, we demonstrated that an antimicrobial peptide (AMP), HPA3PHis, loaded onto a gold nanoparticle-DNA aptamer (AuNP-Apt) conjugate (AuNP-Apt-HPA3PHis) is an effective therapeutic tool against V. vulnificus infection in vivo in mice. HPA3PHis induced bacterial cell death through the disruption of membrane integrity of V. vulnificus. The introduction of AuNP-Apt-HPA3PHis into V. vulnificus-infected HeLa cells dramatically reduced intracellular V. vulnificus by 90%, leading to an increase in the viability of the infected cells. Moreover, when V. vulnificus-infected mice were intravenously injected with AuNP-Apt-HPA3PHis, a complete inhibition of V. vulnificus colonization was observed in the mouse organs, leading to a 100% survival rate among the treated mice, whereas all the control mice died within 40 hours of being infected. Therefore, this study demonstrated the potential of an AMP delivered by AuNP-Apt as an effective and rapid treatment option against infection caused by a major pathogen in humans and aquatic animals.