Burkholderia species are associated with several life-threatening human infections that often result in high morbidity and mortality rates due to their innate resistance to antibiotics. To improve clinical outcomes, new therapies are needed that target conserved, yet unique, Burkholderia pathways. One such pathway is the Burkholderia O-linked protein glycosylation system that is required for virulence in Burkholderia cenocepacia and Burkholderia pseudomallei. This system relies on the O-Glycosylation gene Cluster (ogc), a five gene cluster sufficient and required for the generation of the glycan used for protein glycosylation, and the distally encoded oligosaccharyltransferase, pglL, responsible for the ligation of glycans to glycoproteins. Previous research has shown that the disruption of some genes within the ogc are non-viable, suggesting the perturbation of glycan biosynthesis may provide a novel way to control infections. As such, this work aimed to understand the sensitivity of the Burkholderia O-linked glycosylation system to manipulation and determine how alterations in the expression of proteins in the ogc locus impacts the viability of B. cenocepacia K56-2. Utilising inducible expression systems, we demonstrate that the overexpression of the glycosyltransferases, OgcB and OgcI, results in profound toxicity to B. cenocepacia K56-2. These toxic effects are heightened under the expression of these proteins in genetic backgrounds lacking components of the ogc locus. The removal of predicted active site/s within OgcB and OgcI resulted in improved viability of B. cenocepacia but did not completely abolish toxicity. The overexpression of these glycotransferases also results in alterations in colony and cell morphologies in a reversible manner. We hypothesise that the overexpression of the glycotransferases disrupts the O-linked glycan biosynthesis pathway leading to deleterious impacts due to changes in glycan-lipid intermediates. Thus, these findings provide evidence that the inhibition of the O-linked glycan biosynthesis pathway is extremely sensitive to dysregulation and due to the absence of a close homologue in humans may be an ideal target for the generation of new therapies.