Pseudomonas aeruginosa is a gram-negative, multidrug-resistant, nosocomial pathogen that threatens the lives of immunocompromised patients. A key virulence factor in this pathogen is the siderophore pyoverdine. Due to its extremely high affinity for ferric iron, pyoverdine gives the pathogen a significant advantage over the host in their competition for iron. In addition, pyoverdine can regulate the production of multiple bacterial virulence factors and perturb host mitochondrial homeostasis. To elucidate the regulation of pyoverdine production, we developed a high-throughput genetic screen to identify genes necessary for its biosynthesis. Through this screen, we demonstrated that biofilm formation is necessary for pyoverdine production. Consistent with this result, upstream regulators of biofilm, notably intracellular c-di-GMP, regulated pyoverdine production in a biofilm-dependent manner. Furthermore, inhibiting biofilm formation using 2-amino-5,6-dimethylbenzimidazole was sufficient to attenuate pyoverdine production, suggesting that biofilm inhibitors may have multiple therapeutic benefits in addition to disrupting the pathogen’s main mechanism of antimicrobial resistance.