Pestalotiopsis versicolor is a most destructive fungal pathogen that causes twig blight disease in bayberry. For the last seven years, it is difficult to control this pathogen due to its latent infestation mode and its control through chemical fungicides is environmentally corrosive in addition to being costly. In this study, we reported the fungicidal potential of biologically synthesized zirconium oxide nanoparticles (ZrONPs) against P. versicolor for the first time. The strain used for green synthesis of ZrONPs was taxonomically identified as Enterobacter sp. strain RNT10. The production of ZrONPs in reaction mixture was confirmed through UV–vis spectroscopy analysis. Moreover, FTIR, XRD, SEM and TEM analysis showed the presence of capping proteins and crystalline nature of spherical shaped ZrONPs with particle size ranging from 33 to 75 nm. EDX spectra revealed an elemental profile of ZrONPs comprising of Zr (54.40%) and oxygen (43.49%). Biogenic ZrONPs showed substantial antifungal inhibition zones (25.18 ± 1.52 mm) at 20 μg mL−1 concentration against P. versicolor strain XJ27. Moreover, the treatment of 20 μg mL−1 ZrONPs significantly inhibited twig blight in detached leaf assay. Furthermore, imaging through SEM and TEM showed the adverse effects of ZrONPs against P. versicolor in terms of extracellular leakage of DNA and proteins. Overall, this study suggested that biogenic ZrONPs could substitute chemically synthesized antifungal agents with the specific application towards control of twig blight disease in bayberry.