In aquatic environments such as water distribution systems, Legionella pneumophila persistence is -correlated to the presence of a biofilm. The method described here permits the formation of a monospecies L. pneumophila biofilm in microplates, enabling the screening of multiple parameters. The culture medium used has a low nutrient concentration compared to the classical Buffered Yeast Extract culture medium. Hence, bacterial growth occurs in the sessile phase, allowing the formation of three-dimensional structures.