In the current study, biogenic selenium nanoparticles (Se-NPs) were extracellularly synthesized by Penicillium citrnium. Se-NPs were structurally and morphologically characterized by using analytical techniques such as: X-ray diffraction (XRD), UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR) and transmission electron microscopy (TEM). Maximum UV absorbance was obtained at 255 nm. and the TEM showed 15–40 nm-sized homogeneous spherical Se-NPs. Anticancer potential of the Se-NPs was evaluated against lung cancer (A-459), hepatocellular carcinoma (HepG2), and its cytotoxicity against normal fibroblast cells (WI-38). The Se-NPs have a significant cytotoxic effect (IC50) of 100.2 ± 3.28 µg/mL against HepG2 cells and IC50 of 142.5 ± 6.91 µg/mL against A-549 cells, whereas it has IC50 of 219.75 ± 10.93 µg/mL against Wl-38 cells. In comparison to the untreated HepG2 cells, the treated cells showed a 1.8 and 1.7-fold increase in superoxide dismutase enzyme (SOD) and glutathione (GSH) content, respectively, with a 0.6 fold drop in catalase (CAT) level. The cell apoptosis rates were also assesed using the Annexin V-FITC staining test. In the treated cells, the rates of necrosis, late apoptosis, and early apoptosis were all markedly elevated. The caspase-3 enzyme level of the treated cells was 2.2 folds higher than that of the untreated cells. Flow cytometer showed an arrest in G2/M phase in comparison to untreated cells after treatment of HepG2 with Se-NP. In conclusion, the biogenic Se-NPs exhibited anticancer activity against HepG2 hepatocellular carcinoma cells through enhanced antioxidant production, apoptosis, and cell cycle arrest pathways.