In this study, the antibacterial behavior of differing sized graphene quantum dots (GQDs) was studied. The gram negative bacteria, Escherichia coli (E. coli), were used as the bacteria mode. Different sized GQDs with tunable fluorescent colors were acquired by a gel-filtration method. The size, surface chemistry, and photoluminescence properties of GQDs were characterized, respectively. The viability of GQDs treated bacteria was determined by the standard plate counting method. Moreover, the reactive oxidative species (ROS) level was detected by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) method. The integrality of the bacteria membrane was observed under the scanning electron microscopy (SEM). The experimental results indicated that GQDs demonstrated size-dependent and surface chemistry-dependent antibacterial behaviors. This research provided insightful guidelines in the selection of suitable GQDs for their potential bioapplications.