Bacterial resistance to traditional small molecule antibiotics has led to the need for antimicrobial agents that use an alternative mechanism to kill or inhibit bacterial growth. A promising material for this application is graphene oxide (GO), but the reports of the antibacterial activity have varied.  Also, the mechanisms of antibacterial activity are uncertain, but there are three proposed theories: (i) the edges of the 2D sheets lyse and disrupt the bacterial membrane, (ii) the sheets cover the cell and limit access to vital nutrients, and (iii) GO causes metabolic oxidative stress to the cells. In this study, we investigated the effect of GO’s size on its antimicrobial effects by measuring growth of planktonic Escherichia coli (E. coli) with exposure to GO. We have found nanometer-sized GO (nGO) significantly inhibit the growth of E. coli, but micrometer-sized GO (µGO) did not significantly inhibit E. coli growth, showing GO antibacterial efficacy to be dependent on physicochemical properties. In treating human cells in culture with similar concentrations of GO we find that neither µGO nor nGO had toxic or deleterious impacts on cell function. One major difference between bacteria and human cells is membrane composition and mechanics. To investigate the interaction on a molecular level, we exposed lipid membrane vesicles to µGO and nGO. Altered lipid composition and tension allow us to investigate the membrane impacts in a controlled way. Overall, this knowledge will be informative for the design of future biomaterials to prevent bacterial growth and infections with little impact on human cells.