Bacteria biofilms consisting of surface-attached bacterial communities embedded in an extracellular matrix serve as a defense mechanism for many medically important bacterial species. Exopolysaccharides of partially de-N-acetylated poly-β-D-(1->6)-N-acetyl-glucosamine (dPNAG) are key structural components in the biofilm of many human pathogens. Dispersin B (DspB), a family 20 glycoside hydrolase produced by the Aggregatibacter actinomycetemcomitans, catalyzes the hydrolysis of dPNAG to disrupt biofilm formation leading to its use as an aniti-biofilm agent. Yet little is known of substrate recognition by DspB.Here, we describe the synthesis of two series of PNAG trisaccharide analogs with defined N-acetylation pattern (2.1 – 2.5) or containing glucose moiety (2.32 and 2.33) prepared through an iterative one-pot glycosylation approach and used to profile the activity and substrate preference of DspB (Chapter 2). These studies suggest that DspB hydrolyzes dPNAG polysaccharides via both exo- or endoglycosidase mechanisms and has a substrate preference for cationic substrates at the +2 position of the binding site. Understanding the activity and specificity of DspB provides a valuable guide to develop biocatalyst with improved biofilm dispersal activity. Next, colorimetric (Chapter 3) and fluorogenic (Chapters 4 & 5) PNAG analogs were developed as substrates for high-throughput PNAG glycosidase assay development. PNAG disaccharide probes (3.1 and 5.1) demonstrate exclusive specificity for enzymes capable of hydrolyzing PNAG and monosaccharide analog AMC-GlcNAc (4.1) acts as a general hexosaminidases enzyme substrate. We showed that all the analogs can detect DspB activity in crude E. coli cell lysates, and thus could be applied for functional metagenomic screening to discover novel PNAG glycosidase enzyms. Finally, a series of PNAG triazinyl glycosides (6.1, 6.2 and 6.3) were designed, synthesized and evaluated as affinity labeling reagents for PNAG binding proteins, using a catalytically inactive DspB E184Q mutant as a model PNAG binding protein (Chapter 6). However, only non-specific background signal was observed. In the future, recombinant enzymes or lectins that have higher binding affinity to the PNAG might be used to revisit these labeling results.