Interactions between biofilm cells of Pseudomonas aeruginosa and levofloxacin were studied. P. aeruginosa incubated for 6 days with Teflon sheets formed a biofilm on its surface. Against the biofilm bacteria, levofloxacin at an MIC determined by the standard method for the strain was highly bactericidal whereas gentamicin, ceftazidime, and ciprofloxacin showed no significant killing activity. Levofloxacin, ciprofloxacin, and gentamicin, but not ceftazidime, exhibited killing activity against nongrowing cells of the strain incubated in phosphate buffer. In addition, levofloxacin, ciprofloxacin, and ceftazidime, but not gentamicin, showed the ability to penetrate an agar containing alginate. These findings may explain the efficacy of levofloxacin and the ineffectiveness of gentamicin and ceftazidime against biofilm bacteria; however, the cause of the ineffectiveness of ciprofloxacin still remains to be determined. In experimental pneumonia in guinea pigs, in which the biofilm mode of growth of the strain was observed in the lung, only levofloxacin exhibited substantial therapeutic efficacy. These findings suggest the significant role of levofloxacin in therapy of biofilm bacterium-associated infectious diseases.