Soy isoflavone genistein (Gen) exerts beneficial effects against prostate cancer cells in vitro and in vivo. However, its use as a chemoprevention/therapeutic agent is largely limited due to its low bioavailability. In this study we synthesized two variants of a new delivery system, genistein-gold nanoparticles conjugates Gen@AuNPs1 and Gen@AuNPs2, by an environmentally friendly method, using a dual role of Gen to reduce Au and stabilize the formed AuNPs, with no additional component. The formation of Gen@AuNPs was confirmed via UV-Vis spectroscopy, FTIR, and Raman spectra measurements. The spherical shape and uniform size of Gen@AuNPs1 and Gen@AuNPs2 (10 ± 2 and 23 ± 3 nm, respectively), were determined by transmission electron microscopy. The nano-conjugates also varied in hydrodynamic diameter (65.0 ± 1.7 and 153.0 ± 2.2 nm) but had similar negative zeta potential (-35.0 ± 2.5 and -37.0 ± 1.6 mV), as measured by dynamic light scattering. The Gen loading was estimated to be 46 and 48%, for Gen@AuNPs1 and Gen@AuNPs2, respectively. The antiproliferative activities of GenAuNPs were confirmed by MTT test in vitro on three malignant prostate carcinoma cell lines (PC3, DU 145, and LNCaP), while selectivity toward malignant phenotype was confirmed using non-cancerous MRC-5 cells. Flow cytometric analysis showed that the inhibition on cell proliferation of more potent Gen@AuNPs1 nano-conjugate is comparable with the effects of free Gen. In conclusion, the obtained results, including physicochemical characterization of newly synthesized AuNPs loaded with Gen, cytotoxicity, and IC assessments, indicate their stability and bioactivity as an antioxidant and anti-prostate cancer agent, with low toxicity against human primary cells.