Aims: Cationic steroids like CSA-13 have been designed by analogy with antimicrobial cationic peptides and have bactericidal properties. The purpose of this work was to evaluate the effect of a low concentration (1 mg l(-1)) of CSA-13 on the formation of a biofilm by eight strains of Pseudomonas aeruginosa (four mucoid and four nonmucoid strains) on an inert surface. Method and Results: The biofilm formation was measured with the Crystal Violet method. CSA-13 inhibited the formation of a biofilm by three strains. The zeta potential varied among the strains. The inhibition by the cationic steroid analogue affected the populations of bacteria with the lowest zeta potential. P. aeruginosa bound a fluorescent, more hydrophobic analogue of CSA-13 but there was no correlation between this binding and the inhibition by CSA-13 of biofilm formation. The interaction of CSA-13 with bacteria did not modify their ability to produce rhamnolipids. Conclusions: A low concentration of CSA-13 inhibits the formation of a biofilm by P. aeruginosa through electrostatic interactions and without affecting the production of rhamnolipids. Significance and Impact of the Study: A low, nontoxic concentration of CSA-13 might be beneficial for the prevention of biofilm formation.