Graphene-based materials have attracted a significant attention in constructing hybrid systems for drug delivery with enhanced antimicrobial activities. In our work, we demonstrated the formation of silver nanoparticles (AgNPs) on graphene oxide (GO) using tobramycin (TOB), an aminoglycoside antibiotic, as reducing and decorating agent. The TOB decorated GO AgNPs (TOB-GO-Ag) composite was used as an antibacterial agent against multi-drug resistant Gram-negative E-coli (BL21 DE3). The reversal of surface potential from -30 mV (GO) to +20 mV confirms the successful reduction of GO by TOB. Atomic force microscopy (AFM) and high-resolution transmission electron microscopic (HRTEM) analyses confirmed the formation of uniformly distributed AgNPs on the reduced GO with an approximate particle size of 5 nm. The as-synthesized nanocomposite displayed significant antibacterial activity as compared to pure AgNPs and TOB. The positively charged TOB-GO-Ag interacts with the negatively charged E. coli membrane and inhibit bacterial growth by the antibacterial actions of the released silver, GO and tobramycin from the TOB-GO-Ag composite. The significant loss of bacterial membrane potential from -52 ± 2 mV (control) to -2 ± 1 mV (treated) indicates a severe cell wall damage caused by TOB-GO-Ag composite. Furthermore, fluorescence study also demonstrated a severe membrane disruption in bacterial cells treated with TOB-GO-Ag composite as compared to pure AgNPs and GO. In conclusion, the development of such hybrid systems would help in enhancing the efficacy of available drugs and eradicating the emerging bacterial resistance.