Abstract: | The current research study focuses on biosynthesis of silver nanoparticles (Ag NPs) for the first time from silver acetate employing methanolic root extract of Diospyros assimilis. The UV–Vis absorption spectrum of biologically synthesised nanoparticles displayed a surface plasmon peak at 428 nm indicating the formation of Ag NPs. The influence of metal ion concentration, reaction time and amount of root extract in forming Ag NPs by microscopic and spectral analysis was thoroughly investigated. Structural analysis from transmission electron microscopy confirmed the nature of metallic silver as face‐centered cubic (FCC) crystalline with an average diameter of 17 nm, which correlates with an average crystallite size (19 nm) calculated from X‐ray diffraction analysis. Further, the work was extended for the preliminary examination of antimicrobial activity of biologically synthesised Ag NPs that displayed promising activity against all the tested pathogenic strains.Inspec keywords: antibacterial activity, nanoparticles, silver, particle size, nanofabrication, nanomedicine, biomedical materials, ultraviolet spectra, visible spectra, optical microscopy, surface plasmon resonance, transmission electron microscopy, crystallites, X‐ray diffraction, microorganismsOther keywords: Diospyros assimilis root extract assisted biosynthesised silver nanoparticles, antimicrobial activity, silver acetate, methanolic root extract, UV‐visible absorption spectrum, biologically synthesised nanoparticles, surface plasmon peak, Ag NPs formation, metal ion concentration, reaction time, microscopic analysis, spectral analysis, structural analysis, transmission electron microscopy, metallic silver, FCC crystalline phase, average crystallite size, X‐ray diffraction analysis, pathogenic strains, Ag |