is study explored the possible use of a microbial carboxymethylated sulfated heteroexopolysaccharide (CS-hEPS) as a potential anticancer agent. e investigation was carried out through antioxidant, antifatigue, and antiproliferative activities. Antioxidant potential including scavenging DPPH and hydroxyl radical activities and reducing power was evaluated. Antifatigue activity was determined by assessing the endurance of mice using the forced swimming test. Following 30 days of CS-hEPS oral treatment at different doses, biochemical parameters related to fatigue such as lactic dehydrogenase (LDH), serum urea nitrogen (SUN), and hepatic glycogen (HG) contents were measured. Antitumor activities were investigated against human cancer liver and myelogenous leukemia cells. Results showed that CS-hEPS possesses notable antioxidant, antifatigue, and antitumor effects. CS-hEPS significantly inhibited the proliferation of leukemia (86.6 ± 0.32%) and cancer liver (58.6 ± 0.43%) cells. CS-hEPS are promising natural antioxidant, antifatigue, and antitumor harmless adjuvant materials that could be applied in human cancer therapy.