In the present scenario, resistance to antibiotics has become a threatening situation for public health. To find a solution for this, conjugation of a cationic peptide with silver nanoparticles is emerging as a promising route to attain enhanced antibacterial activity. In this direction, this work reports the synthesis of lysine-based cationic peptide (BP100: NH 2 −KKLFKKILKYL−amide) functionalized silver nanoparticles (BP100@AgNPs). The cationic peptide interacts electrostatically with the silver nanoparticles in an aqueous medium. The developed nanosystem followed green chemistry principles, owing to the use of water and a one-step strategy for the synthesis of peptide functionalized nanoparticles. Moreover, the developed BP100@AgNPs were characterized by UV−vis spectroscopy, zeta potential analysis, FTIR, transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and X-ray diffraction (XRD). In addition to this, in order to have a deeper insight into the interaction between BP100 and silver nanoparticles, NMR analysis of the peptide (BP100) and the developed nanosystem (BP100@AgNPs) was carried out. This was further authenticated by 1D ( 1 H, 13 C) NMR and 2D NMR ( 1 H-COSY (correlation spectroscopy)), 13 C-HSQC (heteronuclear single quantum coherence)). Moreover, the developed BP100@AgNPs were tested for antibacterial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacterial strains. The nanosystem displayed good inhibition with MIC values of 3.60 and 13.20 μg/mL for E. coli and S. aureus, respectively. The destruction in the cellular membrane of the bacterial cells upon treatment with the nanosystem was observed via field emission scanning electron microscopy (FESEM) which confirmed the efficient antibacterial activity of the developed nanosystem. Hence, the synthesized nanosystem displayed considerable potential to be used as an excellent candidate for antibacterial applications.