This study evaluated the biochemical composition and bioactivity of the crude extract of Sargassum dentifolium on the western coast of the Red Sea, Hurghada, Egypt. The biomass contained approximately 25.80% of total carbohydrates, and 8.40% of total protein, while lipids constitute about 2% of the total dry weight which is consistent with the low lipid content typically found in seaweeds. S. dentifolium also produces volatile and non-volatile compounds as part of its metabolic processes. The most abundant volatile compound identified was phytol, which has various industrial and pharmaceutical applications. Saturated fatty acids (SFAs) were the most frequently detected non-volatile compounds, with palmitic acid being the most common. The total phenolic content was 50.6 mg g⁻¹ (5.06%), and the total flavonoid content was 33.9 mg g⁻¹ (3.39%) of the total dry-weight extract. S. dentifolium extract demonstrated significant antioxidant activity, with radical scavenging properties and phenolic content that may contribute to its antioxidant efficacy. It also showed cytotoxic activity against cancer cells, particularly human hepatocellular liver carcinoma (HepG2) and human colon carcinoma (HCT-116) cells, indicating their potential as a source of anti-cancer agents. However, the extract exhibited moderate to weak antiviral activity and limited antimicrobial activity against specific microorganisms. This research could contribute to the development of new functional macroalgae-based food or nutraceutical products with potential health benefits.