The development of new pharmacological strategies that evade bacterial resistance has become a compelling worldwide challenge. Graphene oxide (GO) can represent the nanotechnology answer being economical and easy to produce and to degrade and having multitarget specificity against bacteria. Several groups tried to define the interaction between GO sheets and human pathogens. Unfortunately, controversial results from inhibition to bacterial growth enhancement have been reported. The main difference among all experimental evidence relies on the environmental conditions adopted to study the bacteria–GO interaction. Indeed GO, stable in deionized water, undergoes a rapid and salt-specific DLVO-like aggregation that influences antimicrobial effects. Considering this phenomenon, the interaction of bacteria with GO aggregates having different sizes, morphologies, and surface potential can create a complex scenario that explains the contrasting results reported so far. In this article, we demonstrate that by modulating the GO stability in solution, the antibacterial or growth enhancement effect can be controlled on S. aureus and E. coli. GO at low concentration cuts microorganism membranes and at high concentration forms complexes with pathogens and inhibits or enhances bacterial growth in a surface potential-dependent manner. With the framework defined in this study, the clinical application of GO gets closer, and controversial results in literature can be explained.