This study revealed that a Pseudomonas aeruginosa tonB1 mutant was unable to produce a mature biofilm and showed reduced swarming and twitching motilities compared with the parent strain. The tonB1 mutant was also found to produce significantly lower cell-free and cell-associated levels of the quorum sensing (QS) signal molecule 3-oxo-C12-AHL. Altered biofilm and motility phenotypes were restored to wildtype with the addition of exogenous N-acylhomoserine lactones. These functions were independent of the role of TonB1 in iron uptake. This is the first time that a link has been established between TonB1 activity and QS.