Objective To investigate the effects of ambroxol on the biofilm viability and pristine adhesion of Pseudomonas aeruginosa wild (PAO1) and quorum sensing defective strain (QS, gene deletion of lasI and rhlI). Methods The biofilm was treated by different concentrations (0, 1.875, 3.75mg/ml) of ambroxol. The number of colony was measured with agar plate, multifunction fluorometer was used to measure the fluorescence intensity of PAO1 and QS strains at the bottom of 96-well plate. The adhesion ratio (%) was calculated to determine the effects of ambroxol on bacterial biofilm adhesion. Results Ambroxol treatment reduced the survival rate of the mutant strains compared to that of wild strain, even though the QS strain had increased the adhesion in the presence of ambroxol compared to that of wild strain (P<0.05). Conclusion Ambroxol has a property of significantly antagonizing quorum-sensing system, suggesting that it might be of importance in treatment against chronic Pseudomonasaeruginosainfections.