Previous work has shown that Burkholderia cenocepacia produces the diffusible signal factor (DSF) family signal cis-2-dodecenoic acid (C(12):Delta(2), also known as BDSF), which is involved in the regulation of virulence. In this study, we determined whether C(12):Delta(2) production is conserved in other members of the Burkholderia cepacia complex (Bcc) by using a combination of high-performance liquid chromatography, mass spectrometry, and bioassays. Our results show that five Bcc species are capable of producing C(12):Delta(2) as a sole DSF family signal, while four species produce not only C(12):Delta(2) but also a new DSF family signal, which was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C(12):Delta(2,5)). In addition, we demonstrate that the quorum-sensing signal cis-11-methyl-2-dodecenoic acid (11-Me-C(12):Delta(2)), which was originally identified in Xanthomonas campestris supernatants, is produced by Burkholderia multivorans. It is shown that, similar to 11-Me-C(12):Delta(2) and C(12):Delta(2), the newly identified molecule 11-Me-C(12):Delta(2,5) is a potent signal in the regulation of biofilm formation, the production of virulence factors, and the morphological transition of Candida albicans. These data provide evidence that DSF family molecules are highly conserved bacterial cell-cell communication signals that play key roles in the ecology of the organisms that produce them.