Graphene oxide (GO) is a promising and remarkable nanomaterial that exhibits antimicrobial activity due to its specific surface-interface interactions. In the present work, for the first time, we have reported the antibacterial activity of GO-coated surfaces prepared by two different methods (Hummers' and improved, i.e., GOH and GOI) against bacterial biofilm formation. The bacterial toxicity of the deposited GO-coated surfaces was investigated for both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) models of bacteria. The mechanism of inhibition is different on the coated surface than that in suspension, as determined by measurement of the percentage inhibition of biofilm formation, Ellman's assay, and colony forming unit (CFU) studies. The difference in the nature, degree of oxidative functionalities, and size of the synthesized GO nanoparticles mitigates biofilm formation. To better understand the antimicrobial mechanism of GO when coated on surfaces, we were able to demonstrate that beside reactive oxygen species-mediated oxidative stress, the physical properties of the GO-coated substrate effectively inactivate bacterial cell proliferation, which forms biofilms. Light and atomic force microscopy (AFM) images display a higher inhibition in the proliferation of planktonic cells in Gram-negative bacteria as compared to that in Gram-positive bacteria. The existence of a smooth surface with fewer porous domains in GOI inhibits biofilm formation, as demonstrated by optical microscopy and AFM images. The oxidative stress was found to be lower in the coated surface as compared to that in the suspensions as the latter enables exposure of both a large fraction of the active edges and functionalities of the GO sheets. In suspension, GOH is selective against S. aureus whereas GOI showed inhibition toward E. coli. This study provides new insights to better understand the bactericidal activity of GO-coated surfaces and contributes to the design of graphene-based antimicrobial surface coatings, which will be valuable in biomedical applications. © 2017 American Chemical Society.