Burkholderia multivorans is a gram-negative bacillus that causes opportunistic pulmonary infections in patients having underlying disease. It is hypothesized that the ability to adhere to host tissues is affected by bacterial cell surface properties and most strains are known to elaborate extracellular polysaccharide capsules comprised of disparate biopolymers. The purpose of the present study was to better characterize the cell surface physiology of a type reference strain and seven clinical isolates which represent virulence and colonial phenotypic variants. Microscopic observation, standard macrobroth dilution susceptibility, cell surface hydrophobicity, and biofilm formation analyses were employed to assess pertinent aspects of outer cell surface physiology among strains. The cell surface of the mucoid phenotype was found to be a function of extracellular polysaccharide expression and appeared to facilitate initiation of biofilm production, while being inversely related to cell surface hydrophobic properties. However, the outer cell envelopes of all strains were uniformly permeable to hydrophobic antimicrobial agents as suggested by their uniform minimal inhibitory concentrations. These data support the hypothesis that while extracellular polysaccharide production may affect the ability of B. multivorans to bind to host cells, it does not influence the accessibility of the outer cell surface to nonpolar antimicrobial agents.