Zinc oxide nanoparticles (ZnO NPs) are a famous metal oxide, which is introduced in many daily life applications due to its physiochemical properties. Many parameters can influence the physiochemical properties of ZnO NPs such as pH, temperature, surface chemistry, solvent and precursors. In this work, we aim to investigate the role of hydroxide anionic precursors on the cytotoxicity of ZnO NPs against breast cancer. ZnO NPs were prepared by chemical precipitation using two different hydroxide anionic precursors (Ammonium hydroxide and Sodium hydroxide). X-ray diffraction (XRD) and High-Resolution Transmission Electron Microscope (HRTEM) were employed to study structural properties, while UV-visible spectroscopy was used for measuring the optical properties of the investigated samples. The induced toxic effect was screened using MTT cell proliferation assay. The obtained results confirmed the successful production of ZnO NPs from the two different anionic precursors (Sodium hydroxide and Ammonium hydroxide) using the chemical co-precipitation method. Structural analysis reveals that there is a direct effect of changing the hydroxide anionic precursors on the internal structure and particle size of ZnO NPs. The use of ammonium hydroxide as a hydroxide anionic precursor produces semispherical ZnO NPs with a larger particle size, higher crystallinity and smaller optical bandgap compared with sodium hydroxide. The anticancer activity was measured using MTT assay against breast cancer cells (MCF-7). The obtained results revealed that ZnO NPs synthesized using sodium hydroxide as an anionic hydroxide precursor have a higher activity than ZnO NPs synthesized using ammonium hydroxide.