Biogenic synthesis of nanoparticles provides many advantages over synthetic nanoparticles including clean and non-toxic approaches. Nanoparticle-based application for the development of diagnostics and therapeutics is a promising field that requires further enrichment and investigation. The use of biological systems for the generation of gold nanoparticles (AuNPs) has been extensively studied. The search for a biocompatibility approach for the development of nanoparticles is of great interest since it can provide more targeting and less toxicity. Here, we reported a bio-reductive approach of gold to AuNPs using metabolites extracted from mammalian cells, which provided a simple and efficient way for the synthesis of nanomaterials. AuNPs were more efficiently synthesized by the metabolites extracted from breast cancer (MCF7) and normal fibroblasts (F180) cells when compared to metabolites extracted from cell-free supernatants. The metabolites involved in biogenic synthesis are mainly alcohols and acids. Spectroscopic characterization using UV-visible spectra, morphological characterization using electron microscopy and structural characterization using X-ray diffraction (XRD) confirmed the AuNPs synthesis from mammalian cells metabolites. AuNPs generated from MCF7 cells metabolites showed significant anticancer activities against MCF7 and low toxicity when compared to those generated from F180 cells metabolites. The results reflected the cytotoxic activities of the parent metabolites extracted from MCF7 versus those extracted from F180. Comparative metabolomics analysis indicated that MCF7-generated AuNPs harbored tetratetracontane, octacosane, and cyclotetradecane while those generated from F180 harbored a high percentage of stearic, palmitic, heptadecanoic acid. We related the variation in cytotoxic activities between cell types to the differences in AuNPs-harboring metabolites. The process used in this study to develop the nanoparticles is novel and should have useful future anticancer applications mainly because of proper specific targeting to cancer cells.