Graphene oxide (GO) was synthesized by oxidation of graphite powder using a time-saving modification of Hummers’ method and its antibacterial activity was investigated. Different techniques were applied to characterize the synthesized GO. X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) were used to investigate the crystallinity, morphology, topography and thickness profile of the synthesized graphene oxide, respectively. Fourier-transform infrared spectroscopy (FT-IR) was carried out to investigate the chemical structure and purity of the synthesized sample. Optical properties were investigated by UV-Vis spectroscopy. The antibacterial activity of GO against Escherichia coli (E. coli) was evaluated by the Colony Forming Count (CFU) method. The Results confirmed that the sample had excellent antibacterial activity against Escherichia coli.