This research demonstrates biosynthesis of three different silver nanoparticles (AgNPs) by using tulsi extract and two of its flavonoids, quercetin (QUE) and luteolin (LUT) as reducing agents. The surface morphology, composition and crystallinity are characterized by different techniques. The biomedicinal applicability of synthesized AgNPs were assessed by determining anti-bacterial activity against selected gram-negative (Escherichia coli) and gram-positive (Staphylococcus aureus) bacterial strains. Interaction of biocompatible AgNPs with the HEWL lead to the formation of protein corona. In this regard, the interactions of the synthesized AgNPs with hen egg white lysozyme (HEWL) were investigated by using multi-spectroscopic techniques. Fluorescence studies revealed that upon addition of these AgNPs, quenching of protein fluorescence occurs and it quenching mechanism was static in nature, while the binding affinity of QUE-AgNPs was remarkably higher than Tulsi-AgNPs and LUT-AgNPs. The binding interaction of HEWL with these biocompatible AgNPs is found to be spontaneous and the respective binding distance obtained by using FRET calculations confirms the possibility of non-radiative energy transfer from HEWL to nanoparticles. As indicated by synchronous fluorescence study, a slight decrease in polarity of Trp microenvironment was induced due to the binding with Tulsi-AgNPs while the LUT-AgNPs showed an opposite effect, but the QUE-AgNPs causes no such impacts on the Trp microenvironment. This study provides an informational insight into the polyphenol mediated synthesis of AgNPs, their anti-bacterial activities and the binding interaction between HEWL and biosynthesized AgNPs indicating a new application of nanoparticles in the field of drug transportation and metabolism.