Display omitted •Endophytic fungal supernatant mediated silver nanoparticles (AgNPs).•Formations of AgNPs were confirmed by UV-vis, FTIR, XRD, DLS, EDS, TEM and AFM.•The synthesised AgNPs show good antimicrobial activity. In this study, biological synthesis of silver nanoparticles (AgNPs) from supernatant of endophytic fungus Alternaria sp. isolated from the healthy leaves of Raphanus sativus is studied. The synthesized AgNPs are characterized using UV-vis spectroscopy and Fourier transform-infrared spectroscopy (FTIR). The structural analysis is done by powder X-ray diffraction (XRD) method. The stability of AgNPs is studied by dynamic light scattering (DLS) method. The size and shape of AgNPs are observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM) and found to be spherical with an average particles size of 4–30nm. Further, these AgNPs have been found to be highly toxic against human pathogenic bacteria, suggesting the possibility of using AgNPs as efficient antibacterial agents.