Pseudomonas aeruginosa is known as an opportunistic pathogen whose one of the antibiotic resistance mechanisms includes biofilm formation and virulence factor production. The present study showed that the sub-minimum inhibitory concentration (sub-MIC) of streptomycin inhibited the formation of biofilm and eradicated the established mature biofilm. Streptomycin at sub-MIC was also capable of inhibiting biofilm formation on the urinary catheters. In addition, the sub-MIC of streptomycin attenuated the bacterial virulence properties as confirmed by both phenotypic and gene expression studies. The optimal conditions for streptomycin to perform anti-biofilm and anti-virulence activities were proposed as alkaline TSB media (pH 7.9) at 35 degrees C. However, sub-MIC of streptomycin also exhibited a comparative anti-biofilmefficacy in LB media at similar pH level and temperature. Furthermore, this condition also improved the biofilm inhibition and eradication properties of streptomycin, tobramycin and tetracycline towards the biofilm formed by a clinical isolate of P. aeruginosa. Findings from the present study provide an important insight for further studies on the mechanisms of biofilm inhibition and dispersion of pre-existing biofilm by streptomycin as well as tobramycin and tetracycline under a specific culture environment.