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Microbial biofilms are highly refractory to antimicrobials. The aim of this study was to investigate the use of low-frequency vibration therapy (20-20 kHz) on antibiotic-mediated Pseudomonas aeruginosa biofilm eradication. In screening studies, low-frequency vibrations were applied on model biofilm compositions to identify conditions in which surface standing waves were observed. Alginate surface tension and viscosity were also measured. The effect of vibration on P. aeruginosa biofilms was studied using a standard biofilm assay. Subminimal inhibitory concentrations (sub-MIC) of tobramycin (5 μg/ml) were added to biofilms 3 h prior, during, and immediately after vibration and quantitatively assessed by (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay (XTT) and, qualitatively, by confocal laser scanning microscopy (CLSM). The standing waves occurred at frequencies <1,000 Hz. Biofilms vibrated without sub-MIC tobramycin showed a significantly reduced metabolism compared to untreated controls (p < 0.05). Biofilms treated with tobramycin and vibrated simultaneously (450, 530, 610, and 650 Hz), or vibrated (450 and 650 Hz) then treated with tobramycin subsequently, or vibrated (610 Hz, 650 Hz) after 3 h of tobramycin treatment showed significantly lower metabolism compared to P. aeruginosa biofilm treated with tobramycin alone (p < 0.05). CLSM imaging further confirmed these findings. Low frequency vibrations assisted tobramycin in killing P. aeruginosa biofilms at sub-MIC. Thus, sound waves together with antibiotics are a promising approach in eliminating pathogenic biofilms.