Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in patients with compromised host defense mechanisms, including burn wound victims. In addition to its intrinsic resistance against most antibiotics, P. aeruginosa has the ability to form biofilms adhering to biotic or abiotic surfaces. These factors make treatment of P. aeruginosa infections complicated and demand new therapies and drugs. The flagellum of P. aeruginosa plays an important role in cell–cell and cell–surface interactions during the first stage of biofilm formation. In this study, we describe the selection of monoclonal anti-flagellin single-domain antibodies (VHHs) derived from the Camelid heavy-chain antibody repertoire of a llama immunized with P. aeruginosa antigens. The anti-flagellin VHHs could be produced efficiently in Saccharomyces cerevisiae, and surface plasmon resonance experiments demonstrated that they have apparent affinities in the nanomolar range. Functional screens showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevent biofilm formation in an in vitro assay. These data open doors for the development of novel methods for the prevention of P. aeruginosa-related infections.