Burkholderia pseudomallei is a saprophytic gram negative bacterium that can cause melioidosis in humans and animals upon contact with contaminated environments. The ecosystems inhabited by B. pseudomallei are also populated by organisms that secrete chitin into the local environment. Therefore, the capacity to metabolize this abundant resource is likely to support the survival of bacteria within the niche. Prior to use, chitin must be decomposed into its monosaccharide constituent, GlcNAc, by the combined activity of carbohydrate-active enzymes (CAZymes). The N-acetyl-β-D-hexosaminidase glycoside hydrolases (GHs) catalyse hydrolysis of terminal β1,4-GlcNAc residues from larger chitooligosaccharides. Bacterial N-acetyl-β-D-hexosaminidases have been observed to cleave GlcNAc residues from multiple targets including chitooligosaccharides, glycans, and GlcNAc exopolysaccharides of select bacterial biofilms. This study investigates the function of a putative N-acetyl-β-D-hexosaminidase (bpsl0500) encoded by B. pseudomallei K96243. Bioinformatic analyses confirmed bpsl0500 shared critical catalytic residues and modular components with homologous enzymes within GH family 20. Tertiary structure of bpsl0500 was modelled using Phyre2.0 and observed to superpose with the structurally solved GH20 N-acetyl-β-D-hexosaminidase of Serratia marcescens. Recombinant BPSL0500 enzyme was expressed and purified for biochemical characterization and was subsequently observed to cleave hexosamine residues from both synthetic analogues and the proposed natural substrate, (GlcNAc)2 disaccharide. To explore the biological role of bpsl0500 an unmarked deletion mutant of bpsl0500 was constructed in B. pseudomallei strain K96243 (BpsΔbpsl0500). Deletion of bpsl0500 resulted in the complete loss of chitooligosaccharide metabolism by BpsΔbpsl0500, and significantly decreased N-acetyl-β-D-hexosaminidase activity in liquid culture. The capacity for bpsl0500 activity to degrade biofilm polysaccharide was also investigated by exogenous application of recombinant BPSL0500 to nascent B. pseudomallei biofilms, resulting in a significant decrease in quantifiable biofilm despite the absence of a canonical GlcNAc exopolysaccharide biosynthesis pathway. Combined, these results suggest a dual function for bpsl0500 within chitin rich niches inhabited by B. pseudomallei whereby enzyme activity supports bacterial growth yet potentially interferes with establishment of sessile populations.