Freshwater Nostoc sp. polysaccharides (NSPs) have not been extensively studied, particularly regarding their structural and biological characteristics. This study assessed the extracted NSPs through a multi-faceted characterization approach using 1H and 13C NMR, FTIR spectroscopy, HP-SEC and HPAEC-PAD. The study presented novel insights into the extracted polysaccharide composition, extraction efficiency, and antioxidant activities. NSPs were extracted using two different ranges of heating regimes: cold (25 °C) and hot (95 °C), with hot extraction yielding up to 34.9 % of NSPs compared to 27.6 % from cold extraction. The NSPs were non-sulfated and mainly composed of four monosaccharide residues: (1 → 3)-β-D-mannopyranose-(1 → 4)-β-L-arabinopyranose-(1 → 4)-β-D-glucuronic acid-(1 → 4)-β-D-glucopyranose. Some glucose units were 6-O-methylated, and minor sidechain glycosylation by other monosaccharides was observed. β-D-glucuronic acid was present in higher amounts. The polysaccharides from the inner fluid (IF) fraction exhibited a higher molecular weight (up to 480 kDa) compared to outer layer (OL) fractions. The total phenolic content (TPC: 0.2 ± 0.0–2.8 ± 0.0 %), total sugar (9.1 ± 0.8–67.8 ± 0.4 %), and protein (1.2 ± 0.1–25.9 ± 0.6 %) content were determined. NSPs, especially from cold extraction (1B) exhibited the highest antioxidant activity, have potential applications in the skincare, food, pharmaceutical, and biotechnology industries due to their high yield, unique structural features.