Aminoglycosides are one of the oldest classes of antimicrobials that are being used in current clinical practice, especially on multi-drug resistant Gram-negative pathogenic bacteria. However, the serious side effects at high dosage such as ototoxicity, neuropathy and nephrotoxicity limit their applications in clinical practice. Approaches that potentiate aminoglycoside killing could lower down their effective concentrations to a non-toxic dosage for clinical treatment. In this research, we screened a compound library and identified sanguinarine that acts synergistically with various aminoglycosides. By checkerboard and dynamical killing assay, we found that sanguinarine effectively potentiated aminoglycoside killing on diverse bacterial pathogens, including Escherichia coli, Acinetobacter baumannii, Klebsiella pneumonia and Pseudomonas aeruginosa. The mechanistic studies showed an elevated intracellular ROS and DNA oxidative level in the bacterial cells treated by a combination of sanguinarine with aminoglycosides. Furthermore, an enhanced level of sanguinarine was observed in bacteria in the presence of aminoglycosides, suggesting that aminoglycosides promote the uptake of sanguinarine. Importantly, sanguinarine was shown to promote the elimination of persister cells and established biofilm cells both in vivo and in vitro. Our study provides a novel insight for approaches to lower down the clinical dosages of aminoglycosides.