Bacterial biofilms are thought to aid in the survivability of a variety of intractable infections in humans. Specifically, biofilm production in Pseudomonas aeruginosa has been shown to play a significant role in chronic infection of cystic fibrosis (CF) patients. Unfortunately, no clinically effective inhibitors of biofilm formation are available. A rapid screen of 4509 compounds for nonantibiotic biofilm inhibitors in Pseudomonas aeruginosa PA14 was executed in 384-well plates. Among those compounds, ferric ammonium citrate inhibited biofilm formation in a dose-dependent manner; other iron salts functioned similarly. In addition to biofilm inhibition in static culture, pregrown biofilms could be disrupted and cleared by switching to iron-rich media in flow-chamber experiments. Furthermore, P. aeruginosa strains taken from the sputum of 20 CF patients showed a similar response to elevated iron levels. Previous expression-profiling analyses demonstrated that high levels of iron repress the expression of genes whose products are essential for scavenging iron and that expression of these genes is critical for virulence. Our results, combined with existing transcriptional-profiling data, now indicate that elevated iron concentrations repress the expression of certain genes essential for biofilm production in P. aeruginosa.