The present study was aimed to investigate the composition and contents and the major lipophilic compounds, including the sterols, fatty acids, and tocols of shellfish species. Moreover, to explore the antitumor activity of these lipophilic constituents, their cytotoxicity potentials were determined against five different human cancer cells, including colon carcinoma (HCT116), epithelial melanoma (A2058), glioblastoma multiforme (T98G), lung carcinoma (A549), and adenocarcinoma (HeLa). The results show a significant variation in the contents and composition of lipophilic constituents among the studied species. The highest omega-3 (n-3) polyunsaturated fatty acids (PUFAs) were recorded from arrow squid and pacific oysters, accounting for 53.2% and 53.0% of their total fatty acids, respectively. However, the highest cholesterol content was also recorded in arrow squid (154.4 mg/100 g; 92.6% of total sterols). In contrast, in the Japanese littleneck, Yesso scallop, and common orient clam, cholesterol was just 17.1%, 18.3%, and 18.9% of total sterols, respectively, making them the richest source of non-cholesterol sterols (NCS). Lipids extracted from shellfish species showed ABTS^+•- and DPPH^•-scavenging activities. In the cytotoxicity analysis, lipids extracted from the Argentine red shrimp showed the highest cytotoxicity against glioblastoma multiforme T98G cells, with an IC₅₀ value of 12.3 µg/mL. The composition and cytotoxicity data reported herein may help explore the nutritional and anticancer potentials of shellfish species.