Depending to the WHO, antibiotic resistance and limited anticancer and antimicrobial therapies continue to be serious worldwide health challenges. Current medicines' efficacy suffers by issues such as insufficient solubility, stability, and side effects. To create effective and dependable therapies against antibiotic resistance and robust illnesses, new techniques and strategies are required. Several metal nanoparticles synthesised via green synthesis or chemical synthesise, such as gold (Au), zine (ZnO), and others, have shown promising biological effects against malignancies and a wide spectrum of microbial illnesses caused by multi-drug resistant bacteria. An eco-friendly biosynthetic technique was used to create zinc oxide nanoparticles (ZnO NPs), as well as their antibacterial and anticarcinogenic activities. Extracellular synthesis of nanoparticles made of zinc oxide ZnO nanoparticles was achieved in the current work using the cell filtrate of the endophytic fungus Fusarium chlamydosporum MW341592.1 isolated from healthy leaves of Eucalyptus sideroxylon plant. The nanoparticles were characterised by UV-VIS spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), transition electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDX). The UV-Vis absorption spectra of the produced ZnO NPs showed bands in the UV area at (305) nm. Transmission electron microscopy TEM revealed average sizes of 19.3 nm, while shape revealed spherical like shape. The distinctive pattern of crystalline ZnO NPs was revealed by XRD diffract grams. Furthermore, Biological assay has shown that raising the nanoparticle concentration lowers the number of HCT-116 human colon cancer cells and CACO2 human intestinal cancer cells, as well as antibacterial pathogens Escherichia coli and Pseudomonas aeruginosa.