Fungi-mediated biosynthesis of nanoparticles is a potentially beneficial strategy for large-scale production. In this study, ZnO-NPs were synthesized biogenically using a cell free broth from mycoendophyte Alternaria alternata inhabiting Piper cubeba as a reducer and stabilizer agent. ZnO-NPs appeared to be spherical in transmission electron microscopy, Histogram reveals the nanoparticle size between 20 and 60 nm, and FTIR experiments were employed to analyze the structure, shape, and physicochemical characteristics of the ZnO-NPs. The biosynthesized nanoparticles exhibited an absorption peak at 346 nm in the UV–visible spectrum, which corresponded to the plasmon resonance of ZnO-NPs. The X-ray diffraction spectra of the nanoparticles revealed the formation of crystal planes and hexagonal crystal geometry metal oxides of zinc. In experiments, biosynthesised ZnO-NPs were proven to be exhibit broad spectrum antimicrobial activity. Based on antimicrobial studies bacteria were found to be more susceptible when compare to fungi exhibiting antibacterial activity equal that of co-assayed standard (18–21 mm zone of inhibition). As a result, ZnO-NPs could be mass-produced sustainably, with the potential to be used in a range of biological processes.