Certain environmental conditions can alter the expression of the lipopolysaccharide (LPS) O polysaccharide on the surface of Pseudomonas aeruginosa cells. Changes in O polysaccharide expression influence cell surface characteristics. This study investigated the effect of directly altering O polysaccharide expression on outer membrane vesicle (OMV) production and composition, as well as biofilm formation. P. aeruginosa PAO1 produces two structurally distinct forms of O polysaccharide, the common polysaccharide antigen (CPA) and the O-specific antigen (OSA). Chromosomal knockout mutants of PAO1 that are defective in producing either OSA (∆wbpM) or CPA (∆rmd), or both (∆wbpL) were compared to wildtype cells. It was demonstrated that changes in O polysaccharide expression do not significantly influence OMV biogenesis, but do affect the size and protein sorting of OMVs. In addition, O polysaccharide expression was determined to influence biofilm development, and results from this study highlight the importance of CPA in this process.