Chitosan, obtained from chitin deacetylation, can be hydrolyzed to generate oligomers named Chitooligosaccharides (COS). Lately, COS have attracted considerable interest from several areas due to its biological properties. In this study, the COS was generated by enzymatic hydrolyses using two different hydrolyses times: 1 and 5 minutes, and evaluated the antimicrobial activity of the generated compounds. The 5-minutes hydrolysis showed a superior activity against Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, Candida albicans, Candida glabrata and Candida krusei when compared to the 1- minute hydrolysis. This difference may be related to the different polymerization degree of the oligomers generated in different hydrolysis times. A 2 mg/mL solution of COS produced with 5-minutes hydrolysis could inhibit growth of yeast C. albicans, C. glabrata and C. krusei in 96.53, 97.73 and 96.92%, respectively. It is highlighted that the COS presented antimicrobial activity against all tested microorganisms. Further characterization studies of COS and more complex antimicrobial assays should be performed to understand the relationship between the oligomers structure and their biological properties.