Graphene oxide (GO) is a derivative of graphene nanosheet which is the most promising material of the decade in biomedical research. In particular, it has been known as an antimicrobial nanomaterial with good biocompatibility. In this study, we have synthesized and characterize GO and checked its antimicrobial property against different Gram-negative and Gram-positive multidrug drug resistant (MDR) hospital superbugs grown in solid agar-based nutrient plates with and without human serum through the utilization of agar well diffusion method, live/dead fluorescent staining and genotoxicity analysis. No significant changes in antibacterial activity were found in these two different conditions. We also compare the bactericidal capability of GO with some commonly administered antibiotics and in all cases the degree of inhibition is found to be higher. The data presented here are novel and show that GO is an effective bactericidal agent against different superbugs and can be used as a future antibacterial agent.