Biofilm communities cause many infectious diseases. Biofilms are diverse microbial species found either attached to a surface or aggregated into an extracellular matrix. Bacteria form biofilms as a default mode of growth or as a response mechanism to environmental conditions like stress. As such, biofilm strains are increasingly virulent causing a wide variety of chronic persistent diseases, are typically antibiotic-resistant and known to improve host mortality rate. Most biofilms contain polysaccharides, proteins, extracellular DNA (eDNA), RNA, and water. Determining and quantifying the major components of a biofilm may indicate an appropriate treatment for biofilm eradication. Burkholderia pseudomallei is a Gram-negative, motile bacillus typically found in surface water and/or soil in endemic regions. It is the etiologic agent of melioidosis and is capable of forming both surface adherent and air-liquid interface biofilms (pellicle) in broth cultures. This study evaluates the components of established biofilms using B. pseudomallei and Burkholderia thailandensis, a closely related nonpathogenic species. Using assays, fluorescent dyes and microscopy, we quantified the major components of biofilms produced by five genetically related B. pseudomallei strains and compared them to B. thailandensis E264. Our data show that biofilm produced by the B. pseudomallei 1026b derivatives and B. thailandensis E264 significantly differ. The molecular composition of the surface adherent biofilm is similar to the molecular composition of the air-liquid pellicle. Finally, the eDNA quantity biofilm produced by JW270 which bears a CPS I deletion, is significantly increased in comparison to 1026 and Bp82 biofilm.