This study was conducted to determine the cholesterol and alpha-tocopherol contents of 20 marine fish and four other seafood from the Straits of Malacca. Cholesterol and alphatocopherol contents of the fish and other seafood were determined using high-performance liquid chromatography. The results showed that most of the fish contained low amounts of cholesterol, except sixbar grouper (Epinephelus fasciatus), long-tailed butterfly ray (Gymnura sp.), yellowstripe scad (Selaroides leptolepis), cuttlefish (Sepia officinalis), large-scale tongue sole (Cynoglossus arel), and longtail shad (Hilsa macrura) that contained high amounts of cholesterol (119.39-353.97 mg/100 g wet samples). Indian mackerel (Rastrelliger kanagurta), giant seaperch (Lates calcarifer), prawn (Metapenaeus affinis), and moonfish (Trachinotus blochii) had high alpha-tocopherol contents (462-989 μg/100 g wet sample). Regular consumption of fish and other seafood is highly recommended partly due to the high alphatocopherol content. Due to the high cholesterol in certain types of fish, consumption of the fish fillets of sixbar grouper, long-tailed butterfly ray, yellowstripe scad, cuttlefish, and large scale tongue sole should be < 100 g per day and < 50 per day for longtail shad. Validation of the analytical method also showed a high accuracy and reproducibility of the HPLC method.