We characterized SaHEX, which is a glycoside hydrolase (GH) family 20 exo-β-N-acetylhexosaminidase found in Streptomyces avermitilis. SaHEX 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, SaHEX has potential for use in GlcNAc production and food preservation.