Chitooligosaccharides (COS) are partially hydrolyzed compounds derived from chitosan that exhibit a number of biological activities, including antitumor, antibacterial and antifungal properties. In this work, we examined the cytotoxicity of pure COS and oligomers A, B and C (solutions composed of different amounts of COS) produced by enzymatic hydrolysis using a crude enzyme extract produced by the fungus Metarhrizium anisopliae. The antiproliferative effect of these molecules was analyzed using tumor cell lines (HepG2 and HeLa cells) and in a normal cell line (3T3). The antioxidant activity was analyzed in several in vitro experiments. Glucosamine showed higher toxicity (approximately 92%) to all cell lines studied. However, the oligomers obtained after hydrolysis demonstrated no toxic effects on the normal cells (3T3). Furthermore, we showed that a small amount of other COS can decrease the cytotoxic effect of glucosamine against 3T3 cells, indicating that glucosamine could be used as an antitumor drug in the presence of other COS. In addition, different effects were found in antiproliferative assays, which depended on the COS composition in the oligomers (A, B and C), showing that a combination of them may be essential for developing antineoplastic drugs. Superoxide anion scavenging was the main antioxidant activity demonstrated by the COS and oligomers. This activity was also dependent on the oligomer composition of the chitosan hydrolysates. Further work will identify the ideal proportions of COS and glucosamine for maximizing the effects of these biological activities.