The aim of this study was to investigate the antimicrobial effect of chitooligosaccharides prepared by chitosanase from Pseudomonas CUY8. Antimicrobial activities of different degrees of deacetylation (DD) and polymerization (DP) of chitooligosaccharides against various species of bacteria and fungi were measured. The antimicrobial effects of chitooligosaccharides compared with chitosan and chitosanase were evaluated. Inhibitory diameter of chitooligosaccharides at the concentration of 0.1% with DP 4 was 19 ± 0.20mm, and inhibitory activity with DD 90% was 79± 2.1%, which were higher than other DP and DD, respectively. The results showed that antimicrobial activities of chitooligosaccharides increased with increase of DD, but decreased with increase of DP. Chitooligosaccharides, chitosan and chitosanase all showed significantly stronger antimicrobial activities against bacteria than fungi (p＜0.001). Antimicrobial activities of chitooligosaccharides were significantly higher than that of chitosan (p＜0.05), but insignificantly lower than that of chitosanase (p＞0.05).