The success of Pseudomonas aeruginosa in cystic fibrosis (CF) and other chronic infections is largely attributed to its ability to grow in antibiotic-resistant biofilm communities. This study investigated the effects of limiting iron levels as a strategy for preventing/disrupting P. aeruginosa biofilms. A range of synthetic and naturally occurring iron-chelating agents were examined. Biofilm development by P. aeruginosa strain PAO1 and CF sputum isolates from chronically infected individuals was significantly decreased by iron removal under aerobic atmospheres. CF strains formed poor biofilms under anaerobic conditions. Strain PAO1 was also tested under anaerobic conditions. Biofilm formation by this model strain was almost totally prevented by several of the chelators tested. The ability of synthetic chelators to impair biofilm formation could be reversed by iron addition to cultures, providing evidence that these effective chelating compounds functioned by directly reducing availability of iron to P. aeruginosa. In contrast, the biological chelator lactoferrin demonstrated enhanced anti-biofilm effects as iron supplementation increased. Hence biofilm inhibition by lactoferrin appeared to occur through more complex mechanisms to those of the synthetic chelators. Overall, our results demonstrate the importance of iron availability to biofilms and that iron chelators have potential as adjunct therapies for preventing biofilm development, especially under low oxygen conditions such as encountered in the chronically infected CF lung.