The aim of this study was to synthesize selenium nanoparticles (SeNP) by the process of green synthesis with the addition of olive pomace extract (OPE), which would potentially achieve a synergistic anticancer effect with selenium. SeNPs have the ability to inhibit the growth of cancer cells, while OPE polyphenols show wide spectrum of biological effects, includin anti-cancer efficiency. Yields of proposed green synthesis processes were determined using UV-Vis spectrophotometry, and the particle size distribution and zeta potential were determined using the dynamic- and electrophoretic light scattering methods. The anticancer efficiency was tested on human colon cancer cell lines (Caco-2) and liver tumor cells (HepG2) using the MTT test. A unimodal particle size distribution was obtained, and the average diameters of the obtained nanoparticles ranged from 56.1 nm (for SeNP stabilized with PS and functionalized with OPE (fPSSeNP)) to 181.7 nm (for SeNP stabilized with PVP (PVPSeNP)) which makes these systems particularly suitable for oral administration. PVPSeNP samples have a small absolute value of zeta potential (-5.9 and -10.3 mV), while PSSeNP samples were much more negatively charged (-33 and -30 mV) which may indicate a higher stability of PSSeNP samples. Sodium selenite showed the highest toxicity for the HepG2 cell line, while for the Caco-2 cell line PSSeNP samples were the most toxic. Functionalization of SeNP with OPE reduced and increased the toxicity of SeNP on HepG2 and Caco-2 cell lines, respectively, which also indicates different mechanisms of SeNP toxicity in different cell lines.