Medicinal plants can be used as reducing agents in the preparation of metal nanoparticles by green synthesis because of the chemotherapeutic and anti-infectious properties of natural compounds. Therefore, this paper reports the green synthesis of silver and iron nanoparticles from leaf and flower extracts of Nerium oleander and their capacity as anticancer and antimicrobial agents. Nanoparticle manufacturing and structural characterization of silver and iron nanoparticles are reported. The formation of nanoparticles is characterized by scanning electron microscopy with energy dispersive X-ray spectroscopy, UV-Vis and Fourier transform infrared (FTIR) spectroscopy. Nanoparticles formation was also investigated the surface charge, particle size, and distribution using zeta sizer analysis by DLS. Green synthesis of silver and iron nanoparticles using N. oleander showed different levels of selective cytotoxicity against K562 (human chronic myeloid leukemia cells) in low concentrations and were not cytotoxic to the HUVEC (human umbilical vein endothelial cells) in the same concentrations. Silver nanoparticles showed antibacterial activity against multidrug pathogens, while iron nanoparticles failed to show such activity. Results of the present research demonstrate the potential use of green synthesized nanoparticles in various biomedicine and pharmaceuticals fields in the future.