We report the isolation of insertional mutations to the pstC and pstA genes of the phosphate-specific transport (pst) operon that results in loss of biofilm formation by Pseudomonas aureofaciens PA147-2. Consistent with the known roles of the Pst system in Escherichia coli and Pseudomonas aeruginosa, both P. aureofaciens pst mutants were demonstrated to have defects in inorganic phosphate (P(i)) transport and repression of Pho regulon expression. Subsequently, biofilm formation by the wild type was shown to require a threshold concentration of extracellular P(i). The two-component regulatory pair PhoR/PhoB is responsible for upregulation of Pho regulon expression in response to P(i)-limiting environments. By generating phoR mutants that were unable to express the Pho regulon, we were able to restore biofilm formation by P. aureofaciens in P(i)-limiting conditions. This result suggests that gene(s) within the Pho regulon act to regulate biofilm formation negatively in low-P(i) environments, and that phoR mutations uncouple PA147-2 from such regulatory constraints. Furthermore, the inability of pst mutants to repress Pho regulon expression accounts for their inability to form biofilms in non-limiting P(i) environments. Preliminary evidence suggests that the Pst system is also required for antifungal activity by PA147-2. During phenotypic analysis of pst mutants, we also uncovered novelties in relation to P(i) assimilation and Pho regulon control in P. aureofaciens.