The biofilm component poly-N-acetylglucosamine (PNAG) is an important virulence determinant in medical-device-related infections caused by ESKAPE group pathogens including Gram-positive Staphylococcus aureus and Gram-negative Acinetobacter baumannii. PNAG presentation on bacterial cell surfaces and its accessibility for host interactions are not fully understood. We employed a lectin microarray to examine PNAG surface presentation and interactions on methicillin-sensitive (MSSA) and methicillin-resistant S. aureus (MRSA) and a clinical A. baumannii isolate. Purified PNAG bound to wheatgerm agglutinin (WGA) and succinylated WGA (sWGA) lectins only. PNAG was the main accessible surface component on MSSA but was relatively inaccessible on the A. baumannii surface, where it modulated the presentation of other surface molecules. Carbohydrate microarrays demonstrated similar specificities of S. aureus and A. baumannii for their most intensely binding carbohydrates, including 3′ and 6′sialyllactose, but differences in moderately binding ligands, including blood groups A and B. An N-acetylglucosamine-binding lectin function which binds to PNAG identified on the A. baumannii cell surface may contribute to biofilm structure and PNAG surface presentation on A. baumannii. Overall, these data indicated differences in PNAG presentation and accessibility for interactions on Gram-positive and Gram-negative cell surfaces which may play an important role in biofilm-mediated pathogenesis.