Silver nanoparticles (AgNPs) are toxic to various microbes, but the mechanism of action is not fully understood. The present report explores Azadirachta indica leaf extract as a reducing agent for the rapid biosynthesis of AgNPs. The effects of AgNPs on the growth, glutathione-S-transferase (GST) activity, and total protein concentration in Staphylococcus aureus were investigated, as was its antibacterial activity against seven other bacterial strains. Nanoparticle synthesis was confirmed by the UV-Vis spectrum and colour change of the solution. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential analysis, and infrared spectroscopy were used to characterise the synthesised nanoparticles. The UV-Visible spectrograph showed an absorbance peak at 420 nm. DLS analysis showed an average AgNP size of 159 nm and a Polydispersity Index of 0.373. SEM analysis showed spherical particle shapes, while TEM established an average AgNP size of 7.5 nm. The element analysis profile showed small peaks for calcium, potassium, zinc, chlorine, with the presence of oxygen and silver. AgNPs markedly affected the growth curves and GST activity in treated bacteria, and produced moderate antibacterial activity. Thus AgNPs synthesised from A. indica leaves can interrupt the growth curve and total protein concentration in bacterial cells.