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In this article, the antibiofilm activity and physicochemical properties of graphene oxide (GO) nanoribbons, which have been among the most exciting materials, were studied by measuring the ratio of killed to alive bacteria incubated with these nanomaterials. Our objective was to determine the related physicochemical and antibiofilm properties of graphene oxide nanoribbons. We hypothesized that the physicochemical properties of graphene oxide nanoribbons could affect their antibiofilm activity. A combination of spectroscopic and microscopic measurements of the samples allowed us to determine their physicochemical properties affecting the biofilms. Our work includes information on the surface properties of these materials related to their incubation with the biofilms. The Fourier transform infrared spectroscopy showed the vibrations of OH groups of water molecules adsorbed on graphene oxide nanoribbons. The results show the high antibiofilm activity of the graphene oxide nanoribbons. The fluorescence confocal microscopy revealed that 50 % ± 3 % of the total number of bacteria were killed with these nanomaterials. The incubation of graphene oxide nanoribbons with bacterial biofilms resulted in the appearance of the NO2–, NO3– peaks in the negative mode mass spectrum. The attenuation of the O– and OH– peaks were attributed to the interactions of the samples with the biofilms. Our study gives more evidence of the practical value of graphene oxide nanoribbons in killing bacteria related to their surface physical properties and the potential of these nanomaterials for materials science and biomedical applications.