In the current investigation, a macrofungus was collected from Quaid-i-Azam University Islamabad. The collected fungus was identified as Ganoderma multipileum, and further exploited for the synthesis of nanoparticles using zinc metals. Chemical and morphological characterization of synthetized nanoparticles was through Fourier Transfer InfraRed spectroscopy analysis (FTIR), X-ray diffraction analysis (XRD), Energy dispersive X-ray analysis (EDX), Scanning Electron Microscopy analysis (SEM) and UV-Vis spectrum showed a broad absorption between 350 and 380 nm, which indicates the synthesis of ZnO NPs. ZnONPs have been used in a variety of biomedical studies including such as biocompatibility, antifungal, antileishmanial, and antibacterial studies. abilities, antifungal, antileishmanial, and antibacterial studies. The ZnO nanoparticles showed a strong antibacterial effect against gram-positive (Klesbsilla pneumonia and Staphylococcus aureus) and gram-negative (Eschericia coli and Pseudomonas aeruginosa) bacteria. Furthermore, the ZnO nanoparticles also showed a high antifungal effect against different fungus. The current study concludes that Ganoderma multipileum works as a novel and eco-friendly source for the synthesis of ZnO NPs with prominent biological application that can be further applied in different fields.