Sargassum is the most abundant and diverse macroalgae in Gunungkidul Coast, Indonesia. The use of Sargassum as a cosmetic ingredient in Gunungkidul is limited, despite its antioxidant and antibacterial properties. This study aimed to identify Sargassum based on DNA barcoding and to explore its antioxidant and antimicrobial properties. Three types of Sargassum were collected from the Gunungkidul coast and were identified through DNA barcoding targeting the nuclear ribosomal Internal Transcribed Spacer 2 (ITS2) sequences. Triphasic method of n-heptane/EtOAc/acetonitrile/butane-1-ol/water solvents was employed for extraction. The extract bioactivity was evaluated by testing it against the minimum inhibitory concentration (MIC) of four bacterial species and its IC₅₀ capacity. DNA barcoding identified three confirmed species: S. oligocystum, closely related to S. aquifolium; S. ilicifolium, closely related to S. yinggehaiense; and S. aquifolium, closely related to both S. oligocystum and S. megalocystum. The three phases of triphasic extract were separated into phases: 1 (top), 2 (middle), and 3 (bottom). MIC test against four microbes showed a range of activity from no inhibition to minimal activity. The antioxidant capacity examination of S. ilicifolium and S. aquifolium extracts using IC₅₀ yielded values ranging from strong to very strong. These findings were further supported by the LC-MS dereplication, which revealed the presence of sargachromanols A and cystodione I molecules.