Tambaqui, Colossoma macropomum, is an ideal fish for cultivation in captivity and it is valued in the consumer market. Studies on various aspects of aquaculture, including semen freezing, have been conducted. Despite its many benefits, cryopreservation damages the samples. Thus, to refine the existing protocols, products with antioxidant properties can be added, such as sulfated polysaccharides. This study evaluated the effect of adding sulfated polysaccharides from seaweed (green: Caulerpa cupressoides; red: Acanthophora muscoides and Solieria filiformis) and Nile tilapia skin into the tambaqui semen freezing medium. The resulting polysaccharidic samples were diluted and added to the cryopreservation medium. The semen was then collected, pooled, and subsequently frozen. The pools were diluted and frozen in solutions containing powdered coconut water (ACP-104) and 10% dimethyl sulfoxide (DMSO), and were supplemented with different polysaccharide concentrations (100, 250, 500, or 1000 μg/mL) from seaweed or Nile tilapia skin. Semen cryopreserved in an un-supplemented solution was used as control. After 45 days, the samples were thawed by immersion in a water bath (45 °C/8 s) and the membrane integrity, the morphology and, the sperm motility kinetics were evaluated. There were no differences in sperm kinetics for any of the analyzed parameters that could be linked to the polysaccharide source that was used. For membrane integrity, semen supplemented with sulfated polysaccharides from Nile tilapia skin showed better results (76.3 ± 1.9%) compared to treatments supplemented with A. muscoides (65.0 ± 1.8%), C. cupressoides (66.1 ± 1.7%), or S. filiformis (68.8 ± 1.0%), but there was no difference compared to the control (73.6 ± 2.9%). For morphology, the best results were observed in semen supplemented with C. cupressoides (86.7 ± 0.8%) and S. filiformis (87.7 ± 1.0%) polysaccharides at 100 μg/mL. For the mean path velocity (VAP), semen treated with 250 μg/mL of polysaccharides was significantly faster for A. muscoides (21.1 ± 2.4 μm/s) compared to S. filiformis (15.8 ± 1.7 μm/s). However, at 1000 μg/mL, these two red algae shown to be more efficient than the green algae. In C. cupressoides, 1000 μg/mL of polysaccharides showed lower velocity (13.8 ± 1.5 μm/s) than the other concentrations. In S. filiformis, 100 μg/mL and 1000 μg/mL of polysaccharides did not differ and presented higher VAP than 250 μg/mL and 500 μg/mL. Therefore, the addition of sulfated polysaccharides extracted from aquatic sources to ACP-104 and 10% DMSO solution is safe and has a potential of applicability on C. macropomum semen cryopreservation.