New cancer treatments are desperately needed since the available convetntional cancer drugs have numerous negative consequences. Therefore, the current work attempted to evaluate thecytotoxicproperties of the ink extract (IE) and nidamental gland extract (NGE) from the Egyptian cuttlefish Sepia officinalis on four cancer cell lines: lung carcinoma (A-549), epidermoid carcinoma (A-431), colorectal carcinoma (HCT-116), and prostatic adenocarcinoma (PC-3). Both extracts were characterized through biochemical composition screening via gas chromatography-mass spectrometry (GC-MS) and investigating the levels of proximate composition, phytochemicals, and total antioxidant capacity (TAC). IE and NGE exhibited  cytotoxiceffects by decreasing viable cancer cells number with dose-dependent, with median inhibitory concentrations (IC₅₀) of 517.52 and 427.45µg/ ml against A-549; 511.03 & 262.83µg/ ml against A-431; 480.06 and 220.04µg/ ml against HCT-116 in addition to 372.21 & 242.22µg/ ml against PC-3, respectively. The NGE showed more toxicity toward all tested cancer cell lines than the IE due to higher concentrations of bioactive substances in NGE relative to IE. Moreover, all proximal compositions, phytochemicals, and TAC in NGE were higher than those detected in IE. Hence, the NGE of S. officinalis may be considered a promising cytotoxicagent against cancer cell lines, but more studies are required to explore the action's mechanism.