Burkholderia cenocepacia produces the molecule cis-2-dodecenoic acid (BDSF), which was previously shown to play a role in antagonism against the fungal pathogen Candida albicans by interfering with its morphological transition. In this study, we show that production of BDSF is under stringent transcriptional control and the molecule accumulates in a cell density-dependent manner, typically found with quorum-sensing (QS) signals. B. cenocepacia mutant strain J2315 with a deleted Bcam0581 gene, which encodes an enzyme essential for BDSF production, exhibited a growth defect in minimal medium but not in rich medium, decreased virulence gene expression, and attenuated virulence in a zebrafish infection model. Exogenous addition of BDSF to the mutant rescues virulence gene expression but fails to restore its growth defect in minimal medium. We show that Bcam0581, but not BDSF, is associated with B. cenocepacia ATP biogenesis. We also provide evidence that some of the BDSF-regulated genes are also controlled by the acyl-homoserine-lactone-dependent QS system and are thus coregulated by two cell-to-cell signaling systems. These data demonstrate that in addition to the role in cross-kingdom signal interference, BDSF and its synthase are also important for the virulence and physiology of B. cenocepacia.