Although nanoparticle-tagged antimicrobal peptides have gained considerable importance in recent years, their structure-function correlation has not yet been explored. Here, we have studied the mechanism of action of a designed antimicrobial peptide, VG16KRKP (VARGWKRKCPLFGKGG), delivered via gold nanoparticle tagging against Salmonella infection by combining biological experiments with high- and low-resolution spectroscopic techniques. In comparison with the free VG16KRKP peptide or gold nanoparticle alone, the conjugated variant, Au-VG16KRKP, is non-cytotoxic to eukaryotic cells, but exhibits strong bacteriolytic activity in culture. Au-VG16KRKP can penetrate host epithelial and macrophage cells as well as interact with intracellular S. Typhi LPS under both in vitro and in vivo conditions. Treatment of mice with Au-VG16KRKP post-infection with S. Typhi resulted in reduced intracellular bacterial recovery and highly enhanced protection against S. Typhi challenge. The three-dimensional high resolution structure of nanoparticle conjugated VG16KRKP depicted the generation of a well-separated amphipathic structure with slight aggregation, responsible for the increase of the local concentration of the peptide, thus leading to potent activity. This is the first report on the structural and functional characterization of a nanoparticle conjugated synthetic antimicrobial peptide that can kill intracellular pathogens and eventually protect against S. Typhi challenge in vivo.