Legionella pneumophila is a pathogenic bacterium that forms biofilms in natural and anthropogenic habitats. This feature not only facilitates colonization but also limits the effectiveness of biocides. L. pneumophila was exposed to three sizes of citrate-capped gold nanospheres in both planktonic and biofilm stages. TEM micrographs indicated that gold nanoparticles (AuNPs) adsorbed to the bacterial cell surface, were absorbed into the cells, aggregated within the cells, and integrated into the extrapolymeric matrix of the biofilm. Both 4 and 18 nm, but not 50 nm AuNPs caused an alteration of biofilm morphology. Treatment with 20 nm polystyrene spheres did not induce these changes suggesting that the response was a result of the gold and not just the presence of the nanosphere. The morphological changes observed in the biofilm suggest that aquatic ecosystems may be affected by nanoparticle exposure. This may compromise ecosystem functions such as nutrient cycling facilitated by natural biofilms.