While the cytotoxicity of graphene oxide (GO) has been well established, its bactericidal mechanism, however, has yet to be elucidated to advance GO-based biomedical and environmental applications. In an attempt to better understand the bactericidal action of GO, herein we studied the interactions of GO with Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus cells using physical techniques and chemical probes, respectively. In particular, a novel laser-induced breakdown spectroscopy (LIBS) based elemental fingerprint analysis revealed notable differences between viable and non-viable cells based on the difference in the concentration of trace inorganic elements in complex bacterial systems, which reflect cellular membrane integrity. Lower emission intensities from essential inorganic ions in the GO-treated cells offered explicit evidence on the efflux of intracellular molecules from the bacteria through damaged cell membranes. Furthermore, a detailed structural and morphological investigation of bacterial membrane integrity confirmed GO-induced membrane stress upon direct contact interactions with bacterial cells, resulting in the disruption of cellular membranes. Moreover, the generation of intracellular reactive oxygen species (ROS) in the presence of an added antioxidant underlined the role of GO-mediated oxidative stress in bacterial cell inactivation. Thus, by correlating the changes in the bacterial elemental compositions with the severe morphological alterations and the high ROS production witnessed herein, we propose that the bactericidal mechanism of GO is likely to be the synergy between membrane and oxidative stress towards both tested species. Our findings offer useful guidelines for the future development of GO-based antibacterial surfaces and coatings.