BACKGROUND: Bacteria resist drugs by employing new resistance mechanisms, leading to prolonged infection and complexity in therapeutic prognosis that concordantly decreases drug efficacy. In recent years, nanotechnology has gained immense popularity for the development of drug delivery systems to combat the global pharmaceutical crisis of multi-drug resistance. OBJECTIVE: The present work aimed to focus on the green chemistry-based synthesis of silver nanoparticles (AgNPs) using the phytocompound chlorophyllin to form chlorophyllin precipitated silver nanoparticles (NCHL) and elucidate their application against two pathologically significant bacterial species Escherichia coli and Staphylococcus aureus. METHODS: After optimization of experimental parameters, the physico-chemical properties of the synthesized NCHL were determined using AFM, DLS, XRD, UV-Vis, SPR, and FTIR, respectively. The interaction of NCHL with ct-DNA was assessed using CD spectroscopy. The antimicrobial activity of the synthesized NCHL against Escherichia coli and Staphylococcus aureus was examined by a disc diffusion susceptibility test. RESULTS: The NCHL was ascertained to be ~53.57 nm in size, having a spherical shape, smooth topology, negative zeta potential of -23.94 mV, and PDI value of 0.495. A sharp peak for silver as SPR and an XRD peak depicted the best fit metallic crystal synchronization. FTIR analysis revealed the presence of a similar functional group in NCHL and chlorophyllin (CHL). CD spectroscopy with ct-DNA in the presence of NCHL showed a change in spectral shift of the ct-DNA, indicating strong ct-DNA-NCHL interaction. NCHL successfully inhibited the growth of both bacterial strains, indicating broad spectrum activity of the synthesized nanoparticles. CONCLUSION: The promising results indicated that NCHL could be utilized as a potential therapeutic molecule against Escherichia coli and Staphylococcus aureus infections and help in combating bacterial drug resistance, which is of high medical priority.