Copper is an essential trace mineral that plays an important role in various physiological processes of human body and also possesses excellent antimicrobial properties, however its high dose results in the formation of free-radicals, which can induce cytotoxicity through chromosomal and DNA damage. Therefore, cytotoxicity of colloidal copper nanoparticles (CuNPs) on murine macrophage cell line (RAW 264.7) was studied to understand the correlation between the cytotoxicity and the nanoparticle yield. Three identical sets of CuNPs with similar physical properties having hydrodynamic particle size of 11-14 nm were prepared by chemical reduction method with target yield of 0.2 g, 0.3 g and 0.4 g. CuNPs exhibited dose-dependent (0.001-100 μg/mL) cytotoxicity due to the mitochondrial damage as indicated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. Oxidative stress induced by reactive oxygen species (ROS) in RAW 246.7 macrophage cell lines exposed to CuNPs was the primary cause of observed cytotoxicity in all CuNPs test samples. Morphological changes in cells also indicated strong dose-dependent oxidative damage by CuNPs. IC50 (half maximal inhibitory concentration) values of CuNPs were independent of nanoparticle yield. This suggests that per batch variation in CuNPs yield from 0.2 g to 0.4 g had no negative correlation with their toxicity that makes CuNPs a potential candidate for further development of nanotherapeutics and anticancer drugs.