Burkholderia species are associated with opportunistic infections that are challenging to treat due to innate antimicrobial resistance within this genus. Developing new antimicrobials to control Burkholderia infections represents a major unmet need for at-risk groups, with the Burkholderia O-linked protein glycosylation system being a promising antimicrobial target. Burkholderia protein glycosylation is both highly conserved as well as required for virulence, relying on the O-glycosylation cluster (ogcXABEI) for the stepwise construction and translocation of the trisaccharide β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc. Previous work has suggested that blocking late-stage steps in this pathway may be conditionally lethal, yet this has not been confirmed. Using Burkholderia cenocepacia (Bc) as a model, we have investigated the potential of perturbing O-linked glycosylation biosynthesis as a potential anti-microbial target. By developing rhamnose-inducible strains that allow the control of glycosylation initiation, we show that blocking late-stage glycan biosynthesis by the deletion of ogcA and ogcX leads to marked phenotypic effects, which are alleviated by reintroducing ogcA and ogcX. Proteomic analysis supports the loss of ogcA/ogcX blocks glycosylation and drives dramatic effects on the membrane proteome supporting the fouling of the undecaprenyl pool within these mutants. Consistent with this model, phenotypic assays revealed that ΔogcA and ΔogcX exhibit increased sensitivity to membrane stressors as well as marked changes in membrane permeability. Finally, we show that overexpression of early-stage glycan biosynthesis steps (ogcI and ogcB) drives detrimental effects, supporting the sensitivity of the undecaprenyl pool to fouling, as well as that ogcA/ogcX mutant toxicity can be suppressed by the overexpression of undecaprenyl pyrophosphate synthase, suggesting that UndP sequestration may contribute to the observed effects. These findings demonstrate that completion and transport of O-linked glycans is critical for both glycosylation and Bc viability and could be exploited as an anti-microbial target to combat Burkholderia infections.