Burkholderia pseudomallei, a motile and rod Gram-negative bacterium, is the causative agent of melioidosis. The bacterium is an intracellular pathogen and that motility is generally crucial for their survival in a natural environment and for systemic infection inside a host. We report here a role of B. pseudomallei polyphosphate kinase in virulence, such as an oxidative stress response, motilities and biofilm formation. The polyphosphate kinase (ppk) mutant is susceptible to hydrogen peroxide in an oxidative stress condition, unable to perform swimming, swarming motilities, and has lower density biofilm forming capacity than the wild-type strain. We also demonstrated that both polyphosphate kinase and motile flagella are essential and independently involved in biofilm formation. The B. pseudomallei flagellin (fliC) mutant and B. mallei, a nonmotile species, are shown to produce higher density biofilm formation than the ppk mutant, but less than wild type B. pseudomallei.