Human infection by bacteria of the genus Legionella most often result in the pneumonia known as Legionnaires Disease. Legionella is found as a resident of adherent biofilms in man-made water systems. Disinfection efforts to prevent Legionella infections require a better understanding of the structures that promote Legionella surface attachment and biofilm colonization. Various enzymatic treatments, including multiple carbohydrate-targeting mixtures, failed to disrupt Legionella biofilms, despite the presence of carbohydrates in the biofilms as shown by biochemical methods and concanavalin-A lectin staining. Moreover, Legionella biofilms contained amyloids as detected by three microscopic staining methods (congo red, thioflavin T, and the amyloid-specific antibody WO2). Amyloid structures were seen in biofilms of both L. pneumophila and L. longbeachae, the two Legionella species most associated with human infection. Inhibition of amyloid assembly by congo red and thioflavin T limited both self-aggregation and surface attachment of L. pneumophila, indicating that functional amyloid structures have a key role in initial biofilm formation by these pathogenic bacteria.