In order to promote the biological applications of graphene based materials, great exploration for nano-scale preparation of graphene oxide (GO) has been made with a novel facile processing method and low cost. For these reasons this method has been carried out in the following study. The GO nanosheets have been prepared depending on Hummer's method, which represents an easy and efficient technique since 1958. Alternations have been done to simplify this method in order to prepare the GO with a novel way without a need to add NaNO 3. Also this preparation has been achieved at room temperature without a need to water bath at temperatures of 35 C and 98 C. Structural and morphological properties and contents of the synthesis nanosheets were characterized by using XRD, SEM, and EDS techniques, respectively. The purpose of this work is to evaluate the antibacterial activity of GO nansheets against Gram-negative and Gram-positive bacteria of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively. Bacteriological test were achieved by colony forming units (CFUs) assay. CFUs assay has ensured potential of GO as an anti-infective agent for controlling the growth of two spices of bacteria. The results have showed that Gram-positive were more effective by nanosheets than Gram-negative.