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In the present article, we have developed an eco-friendly, phytosynthetic, cost-effective, and straightforward method for the synthesis of ZnO nanoparticles using leaf extracts of Juglans Regia (JR) as green reducing agents. The powder X-ray diffraction pattern revealed the high crystalline nature of synthesised ZnO nanoparticles also rivalled with ICSD no. 98–009-4004. The SEM images of the samples showed the spherical and irregularly distributed rod and plate-like structures. The EDS spectrum filed only Zn and O peaks revealed that the sample has good purity in ZnO nanoparticles. The HRTEM analysis explained the formed ZnO nanoparticles by biogenic reduction were hexagonal shape with definite edges and the average grain size was obtained as 33.18 ± 6.2 nm for the 50JR sample. The d-spacings of fringes and SAED pattern got also rivalled with XRD d-spacings. According to the UV-Visible analysis, the values of peak absorbance wavelength decreased (blue shift) with the increase in the leaf extract concentrations. The bandgap values were calculated in the range of 2.74–3.17 eV. The Fourier Transform Infra-Red study explicated the biomolecules for the reduction of nanoparticles and also ZnO bonding. These results also proved the presence of ZnO nanoparticles synthesised using Juglans Regia extract. Regarding the antimicrobial effects of ZnO NPs, ZnO NP which was using 50 mL JR extracts was detected as highly efficient nanoparticles against test microorganisms. In particular, the highest antimicrobial activity was measured with 15.66 ± 1.15 mm against Candida albicans 10,231 and revealed data compared with fluconazole, as reference antifungal, recorded 69% antimicrobial index. The findings demonstrated that ZnO NPs can be evaluated for use in biomedical applications. [ABSTRACT FROM AUTHOR]