Gold nanoparticles (AuNPs) have recently emerged as prominent vehicles for many biomedical applications from sensing to delivery. The relevant literature contains conflicting data about the effects of AuNPs on living cells. The aim of present study is the synthesis and characterization of AuNPs at nanoscale, tracking their cellular localization and determining their effects on cell viability, migration and angiogenesis. Within this scope, 20 nm AuNPs were synthesized and characterized using various spectrometric techniques to determine their size, shape and surface properties such as charge and texture. Two main cell types including mouse fibroblast (L929) and human cervix adenocarcinoma (HeLa) were used in the study to compare the biological effects of colloidal gold on both non-cancer and cancer cells. AuNPs were allowed to interact with HeLa cells to determine their intracellular localization. AuNPs were mainly attached to the cell membrane/membranous compartments and to be captured in small amounts in cytoplasmic vacuoles or to be distributed freely in the cytosol. Scratch assay results showed that AuNPs reduced cancer cell migration especially at increasing concentrations. According to the chick chorioallantoic membrane assay, AuNPs exhibited strong anti-angiogenetic properties and can inhibit vascularization during angiogenesis. In addition, the MTT assay confirmed that AuNP-treated cells caused concentration dependent cytotoxic effects on both cell types. As a result, AuNPs not only have inhibitory effects on cancer cells, but also possess antiangiogenic activity, thus making them a multipotent agent for cancer therapy. (c) 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1708-1721, 2018.