The present study was carried out to determine the antitumor and antioxidant activities of the seaweed Durvillaea antarctica. Extraction and purification of polysaccharides from D. antarctica were performed. They were characterized by FT-IR and GC–MS, identifying isomers of arabinose, fucose, mannose, and galactose. The antioxidant capacity of polysaccharides was analyzed using the ABTS method (14.3 ± 0.5 μmol TE g−1 PS) and the DPPH method (21.82 ± 0.32 μmol TE g−1 PS). The antitumor capacity of polysaccharides was studied by MTT colorimetric assays in human leukemia, colon, breast, and lung cancer cell lines, obtaining the lowest IC50 in colon cancer (19.99 μg mL−1). In the line of healthy human gingival fibroblasts (HGF-1), an IC50 of 444.39 μg mL−1 was obtained. Flow cytometry in the HL60 cell line showed that polysaccharides at concentrations higher than IC50 inhibited cell proliferation, demonstrating a possible antitumor capacity in vitro. In the proteomic analysis with HGF-1, nine proteins involved in different biological processes were identified. In conclusion, polysaccharides from D. antarctica could be considered powerful nutraceuticals, mainly against colon cancer.