This study investigates the green synthesis of AgNPs from 1 mM aqueous AgNO3 using 10% leaf extract of Alstonia scholaris (Chhatim) for its wide antibacterial and medicinal properties. The synthesized AgNPs were duly characterized by UV-vis (UV-vis) spectrophotometry, dynamic light scattering, field emission scanning electron microscopy, transmission electron microscopy, energy-dispersive analysis of X-rays spectroscopy, and fourier transform infrared spectrophotometry. Their antibacterial property was tested against Escherichia coil (ATCC 25922), and minimum inhibitory concentrations of 0.08 nM of AgNPs were obtained, which suggests improved therapeutic efficacy. We report the interaction of human serum albumin (HSA) with this nanoparticle, and this interaction was studied by UV-vis, fluorescence, and circular dichroism spectroscopies and zeta potential measurement at room temperature. It was found that the AgNPs form a complex with HSA, which may cause the slightest change in the conformation of HSA. The calculated values of Stem-Volmer quenching constant, binding constant, and binding distance were 1.82 x 10(7) M-1, 1.58 x 10(7) M-1, and 3.68 nm, respectively. Therefore, in future, the present study may provide useful information to design a better antibacterial compound by using green synthesized nanoparticles with fewer side effects.