Exopolysaccharides consisting of partially de-N-acetylated poly-β-d-(1→6)-N-acetyl-glucosamine (dPNAG) are key structural components of the biofilm extracellular polymeric substance of both Gram-positive and Gram-negative human pathogens. De-N-acetylation is required for the proper assembly and function of dPNAG in biofilm development suggesting that different patterns of deacetylation may be preferentially recognized by proteins that interact with dPNAG, such as Dispersin B (DspB). The enzymatic degradation of dPNAG by the Aggregatibacter actinomycetemcomitans native β-hexosaminidase enzyme DspB plays a role in biofilm dispersal. To test the role of substrate de-N-acetylation on substrate recognition by DspB, we applied an efficient preactivation-based one-pot glycosylation approach to prepare a panel of dPNAG trisaccharide analogs with defined acetylation patterns. These analogs served as effective DspB substrates, and the rate of hydrolysis was dependent on the specific substrate de-N-acetylation pattern, with glucosamine (GlcN) located +2 from the site of cleavage being preferentially hydrolyzed. The product distributions support a primarily exoglycosidic cleavage activity following a substrate assisted cleavage mechanism, with the exception of substrates containing a nonreducing GlcN that were cleaved endo leading to the exclusive formation of a nonreducing disaccharide product. These observations provide critical insight into the substrate specificity of dPNAG specific glycosidase that can help guide their design as biocatalysts.