Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, often diagnosed at advanced stages when curative options are limited and conventional therapies like sorafenib show modest efficacy and significant toxicity. This highlights the urgent need for innovative, targeted treatments with improved selectivity and safety. Snake venoms are a rich source of bioactive molecules with therapeutic potential, particularly in the field of oncology. This study explores the anticancer properties of Moroccan Naja haje venom and its purified fractions against HCC using both conventional monolayer (2D) cultures and physiologically relevant multicellular tumor spheroid (3D) models. Crude venom was fractionated by gel filtration and RP-HPLC, and the resulting components were tested for cytotoxicity on HepG2 and Huh7.5 liver cancer cell lines. Their efficacy was further evaluated in 3D spheroids co-culturing cancer cells with human fibroblasts (WI38), hepatic stellate cells (LX2), and endothelial cells (HUVECs). Normal hepatocytes (Fa2N-4) were used to assess off-target toxicity. Several fractions, particularly Fraction 7 and its subfraction 7d, significantly inhibited cancer cell proliferation and reduced spheroid size, with minimal toxicity to normal liver cells,demonstrating greater selectivity than sorafenib. Proteomic analysis identified cytotoxins 2, 5, and 10 as key components responsible for this activity. These findings underscore the potential of Naja haje venom-derived peptides as promising candidates for selective HCC therapy,warranting further investigation into their mechanisms of action and therapeutic development.