We characterized Sa HEX, which is a glycoside hydrolase (GH) family 20 exo-β- N -acetylhexosaminidase found in Streptomyces avermitilis . Sa HEX exolytically hydrolyzed chitin oligosaccharides from their non-reducing ends, and yielded N -acetylglucosamine (GlcNAc) as the end product. According to the initial rate of substrate hydrolysis, the rates of (GlcNAc)₃ and (GlcNAc)₅ hydrolysis were greater than the rates for the other oligosaccharides. The enzyme exhibited antifungal activity against Aspergillus niger , which was probably due to hydrolytic activity with regard to chitin in the hyphal tips. Therefore, Sa HEX has potential for use in GlcNAc production and food preservation.