The influence of azithromycin on biofilm formation by Pseudomonas aeruginosa, a cause of refractory chronic respiratory tract infection, was investigated. Alginic acid produced by a mucoid strain of P. aeruginosa was quantified by high-performance liquid chromatography from colonies growing on an agar medium. Polysaccharides in the biofilm formed on silicon chips by a nonmucoid strain were determined by a tryptophan reaction. The effect of azithromycin was examined at concentrations below the minimum inhibitory concentration (sub-MIC) for each strain. Azithromycin significantly inhibited the production of alginic acid from the mucoid strain at ≥ 1/256 MIC, and the production of exopolysaccharides from the nonmucoid strain at ≥ 1/16 MIC. The inhibition of biofilm formation by azithromycin was also observed by scanning electron microscopy. These findings suggest that azithromycin inhibits biofilm formation by P. aeruginosa at concentrations well below the MIC.