Bacterial biofilms are problematic in natural and anthropogenic environments, and they confer protective properties on their constituent cells, making them difficult to treat with conventional antibiotics. Antibiofilm strategies, therefore, represent a promising direction of research for treating biofilm infections. Natural autodispersal and interspecies dispersal signaling pathways provide insight into cell-cell communication mechanisms, species dynamics in mixed communities, and potential targets for infection therapies. Here, we describe a novel interspecies dispersal signaling pathway between Pseudomonas aeruginosa and Escherichia coli. E. coli biofilms disperse in response to compounds in P. aeruginosa culture supernatant. Two components of the P. aeruginosa Las and Rhl quorum sensing systems, N-(3-oxo-dodecanoyl) homoserine lactone (3oxoC12HSL) and rhamnolipids, are found to act cooperatively to disperse E. coli biofilms. Our results indicate that rhamnolipids do not affect growth, biofilm development, or dispersal in E. coli but instead complement 3oxoC12HSL signaling by inducing selective permeability of the E. coli membrane. The increased target cell permeability is consistent with rhamnolipid-mediated removal of lipopolysaccharide from E. coli membranes and appears to selectively increase the permeability of lipophilic acyl homoserine lactones. This work suggests that rhamnolipids play a critical role in P. aeruginosa-E. coli interspecies signaling. Rhamnolipids and other biosurfactants may have similar effects in other intra- and interspecies chemical signaling pathways.