Understanding the interactions of graphene oxide (GO) with biological membranes is crucial for the evaluation of GO’s health and environmental impacts, its bactericidal activity, and to advance graphene-based biological and environmental applications. In an effort to understand graphene-induced bacterial inactivation, we studied the interaction of GO with bacterial (Escherichia coli) cell membranes using atomic force microscopy (AFM). Toward this goal, we devised a polydopamine-assisted experimental protocol to functionalize an AFM probe with GO nanosheets, and used AFM-based force spectroscopy to measure cell membrane–GO interaction forces. Our results show that GO–cell interactions are predominantly repulsive, with only sporadic adhesion forces being measured upon probe pull-off, which we attribute to lipopolysaccharide bridging. We provide evidence of the acellular oxidation of glutathione by GO, underscoring the role of oxidative pathways in GO-mediated bacterial cell inactivation. Our force spectroscopy results suggest that physicochemical interactions do not underlie the primary mode of action of GO in bacteria.