Green synthesis techniques have recently become more popular due to the expanding interest in nanotechnology and the need for ecologically friendly synthesis processes. This work examines the environment friendly production of Zinc Oxide Nanoparticles (ZnO NPs) by a microwave-assisted technique, utilizing Pistia Stratiotes leaf extract as a reducing agent. The optical and structural properties of the produced ZnO NPs were analyzed using UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and X-ray Diffraction (XRD). The findings indicated that the ZnO NPs displayed shapes consisting spherical, flower and sheet, with an average size of 35 nm which was verified by SEM and TEM. The XRD examination confirmed the presence of a hexagonal wurtzite crystalline structure, while the FTIR analysis identified a distinct peak at 578 cm-1, which indicates the stretching mode of Zn-O bonds. The antibacterial and antifungal properties of the substance were evaluated against Staphylococcus Aureus, Escherichia Coli, and Candida Albicans. The results demonstrated notable effectiveness, as indicated by inhibition zones measuring 16-20.4 mm, 17-21.3 mm, and 13-17.2 mm correspondingly. In addition, the ZnO NPs exhibited specific anti-cancer effects on SK-MEL-28 melanoma cell lines, with an IC50 value of 51.05 µg/ml, suggesting potential uses in therapy. This study demonstrates the feasibility of using microwave-assisted green synthesis to create ZnO NPs with favorable characteristics for biomedical purposes, with a particular focus on sustainable manufacturing of nanomaterials. The results indicate that these ZnO NPs show great potential for application in antibacterial, antifungal, and anticancer therapies, leading to developments in the field of medical nanotechnology.