Marine algae polysaccharides (MAP) were extracted from brown macroalgae Sargassum ilicifolium, which has been recognized as a rich source of sulfated polysaccharide, fucoidan. Three techniques including hydroethanolic extraction or conventional extraction (CE), microwave-assisted extraction (MAE) and subcritical water extraction (SWE) were applied. Extraction yield (EY), total phenol content (TPC), sulfate content, Nuclear magnetic resonance (NMR) analysis, antioxidant activity, monosaccharide composition, molecular weight (MW) distribution, antimicrobial, antiviral, and anticancer activities were assessed. Results showed that the MAP-SWE indicated higher EY (7.86 %) compared to MAP-MAE (6.60 %) and MAP-CE (5.97 %). The highest TPC and sulfate content and subsequently the highest antioxidant potential were obtained for MAP-SWE, followed by MAP-MAE, and MAP-CE. The highest fucose unit which was substituted with sulfate groups was obtained in MAP-SWE (268.48 nmol), followed by MAP-MAE (244.45 nmol), and MAP-CE (112.46 nmol). Some signals of C-NMR and H-NMR indicated higher fucoidan EY for MAP-SWE. The smallest MW was attained for MAP-SWE (517.74 kDa), followed by MAP-MAE (814.20 kDa), and MAP-CE (1188.65 kDa). The MAP-SWE were more efficient in antimicrobial activity while indicated 11 Staphylococcus aureus colonies at 100 μg/mL and 2 Escherichia coli colonies at 400 μg/mL. The extracted MAPs were more efficient on Gram-positive bacteria. Regarding the antiviral and anticancer activities, the strongest values were obtained for MAP-SWE likely due to its higher sulfate and TPC which are considered as crucial factors in biological activities. Consequently, SWE was more efficient in extraction of MAPs, rendering well-deserved properties in terms of biological activities.