Seagrasses are an excellent and potential bioresource to discover new natural bioactive compounds such as antioxidants that have beneficial effects on health. Natural antioxidants have many functions in biological systems, primarily for defense against oxidation which produces free radicals in food, chemicals, and living systems. The present study was conducted to determine the preliminary phytochemical analysis, antioxidant and cytotoxicity activity of hydroalcoholic extract of Enhalus acoroides. The hydroalcoholic extract were evaluated for total phenolic and total flavonoids contents. The antioxidant activity of hydroalcoholic extract of Enhalus acoroides was assessed by different scavenging assays. The present study revealed the presence of major phytochemicals like alkaloids, phenols, flavonoids, steroids, tannins and terpenoids. Quantitative analysis of the total phenols was found to be 45.95 mgGAE/g and the total flavonoids were found to be 33.42 mgQU/g in the aqueous methanolic extract. This study aimed to discover new natural antioxidant agents, Enhalus acoroides was evaluated for phytochemical constituents and the antioxidant activity was assessed. The information of secondary metabolites from E. acoroides will contribute to further research in determination of antioxidant, and anticancer of E. acoroides, as a promising marine drug candidate from India.