The current study reports on polyphenols profile of pomegranate leaves (PL) Punica granatum grown in Egypt and exhibiting their role in development of an eco-friendly method of green synthesis of silver nanoparticles (AgNPs). PL aqueous alcohol extract was fractionated, the major phenolic compound was isolated from the polyphenols rich fraction (ethyl acetate fraction) and identified by conventional and spectroscopic methods of analysis as ellagic acid. Furthermore, the fraction was standardized and analysed using UPLC-PDA-UV and LC-MS-MS techniques revealing tentative identification of 23 polyphenolic compounds, quantifying ellagic acid as 43.14 ± 0.57 μg/mg of the fraction. AgNPs were successfully synthesized with the aid of polyphenols rich fraction. This is the first report revealing the systematic optimization of the green synthesis process using various independent variables. AgNPs were characterized by adopting UV-Vis spectroscopy, FTIR, XRD, and SEM, which revealed strong SPR band within average of λ max 425 nm and polygonal shaped nanoparticles of 26.22 nm size, respectively. The antimicrobial efficacies of AgNPs and polyphenols rich fraction were tested against Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, and Sarcina lutea), Gram-negative bacteria (Salmonella paratyphi, Escherichia coli, and Pseudomonas aeruginosa) and fungi (Candida albicans). AgNPs showed a concentration-dependent activity against all the tested microorganisms.