This study focuses on the extracellular green synthesis of zinc oxide nanoparticles (ZnO NPs) using cell filtrate from yeast Rhodotorula glutinis and investigate the anticancer activity of these nanoparticles and compare it with the cytotoxicity effect of its biomass extraction on a breast cancer cell line. The optical properties of the nanoparticles were analyzed using UV—vis spectroscopy which showed a distinct absorption peak at (375.50) nm, while Fourier transform infrared spectroscopy (FT-IR) was proofed presence of characteristic functional groups belongs to ZnO, C=C, C=O, C-H and OH. The data obtained from the MTT assay indicate that the ZnO NPs exhibit high toxicity and significantly decrease cell viability in the breast cancer cell line compared to the biomass extraction and untreated cell line.