<p>Chitinolytic enzymes are capable of hydrolyzing the cell walls of pathogenic fungi, disrupting the exoskeletons of insect pests, and producing N-acetyl-β-D-glucosamine oligomers or monomers. In this study, a new β-N-acetylhexosaminidase gene encoding an enzyme from glycoside hydrolase family 20 (GH 20) was identified from the biocontrol fungus <i>Chaetomium globosum</i> W7. The corresponding protein was designated CgNagase20 and was heterologously expressed in <i>Escherichia coli</i>. The molecular mass of the protein was approximately 66.7&#xa0;kDa, and this enzyme exhibited maximal activity at 55&#xa0;°C and pH 4.0. Heavy metal ions, particularly Fe³⁺, Ag⁺, and Hg²⁺, were found to strongly inhibit CgNagase20 activity. CgNagase20 exhibits typical substrate specificity of β-N-acetylhexosaminidase (EC 3.2.1.52), acting on N-acetylglucosides and N-acetylgalactosides, with monosaccharides as the sole end products. The Vmax and Km values for CgNagase20 when acting on 4-nitrophenyl N-acetyl-β-D-glucosaminide (pNP-βGlcNAc) were calculated to be 118.85 µmol/min/mg protein and 1.59 mM, respectively. Additionally, CgNagase20 demonstrated antifungal activity, significantly inhibiting spore germination and hyphal growth of <i>Fusarium sporotrichioides</i>, the causative agent of potato dry rot. In conclusion, CgNagase20 represents the first β-N-acetylhexosaminidase identified and characterized from <i>C. globosum</i>, with potential applications in both the industrial and agricultural sectors.</p>