AbstractReduced graphene oxide (rGO) is a promising antibacterial material, the efficacy of which can be further enhanced by the addition of silver nanoparticles (nAg). In this study, the mechanisms of antibacterial activity of rGO–nAg nanocomposite against several important human pathogenic multi-drug resistant bacteria, namely Gram-positive coccal Staphylococcus aureus and Gram-negative rod-shaped Escherichia coli and Proteus mirabilis are investigated. At the same concentration (100 µg/ml), rGO–nAg nanocomposite was significantly more effective against all three pathogens than either rGO or nAg. The nanocomposite was equally active against P. mirabilis and S. aureus as systemic antibiotic nitrofurantoin, and significantly more effective against E. coli. Importantly, the inhibition was much faster in the case of rGO–nAg nanocomposite compared to nitrofurantoin, attributed to the synergistic effects of rGO–nAg mediated contact killing and oxidative stress. This study may provide new insights for the better understanding of antibacterial actions of rGO–nAg nanocomposite and for the better designing of graphene-based antibiotics or other biomedical applications.