Featured Application The antibacterial activities of graphene nanostructures (GrNs) grown onto glass (G) and nickel (Ni) substrates against E. coli and S. aureus have been investigated. The interactions of bacteria with GrNs synthesized onto various substrates (G and Ni) by using different deposition methods (CVD and PECVD) have been examined and the evaluation of these interactions according to the number of layers of graphene and different RF powers has been done. As graphene is transparent, flexible, biocompatible, and thermally stable, the findings may offer new viewpoints both for the better interpreting of the antibacterial activity of GrNs and for the better designing of graphene-based food-packing and biomedical device applications. The antibacterial activity of graphene nanostructures (GrNs) on glass (G) and nickel (Ni) substrates against Escherichia coli ATCC 35218 (Gram-negative) and Staphylococcus aureus ATCC 25923 (Gram-positive) has been researched in this study. GrNs have been synthesized via two different methods, namely, chemical vapor deposition (CVD) and plasma enhanced chemical vapor deposition (PECVD). While the antibacterial effect of CVD-grown graphene nanosheets has been examined according to the number of layers (monolayer/1-2 layers/2-3 layers), the effect of PECVD grown Gr nanowalls on G substrates has been also analyzed at 100, 150, and 200 W radio frequency (RF) powers. For CVD-grown graphene nanosheets, as the number of layers of graphene nanosheets decreased, the cell viability (%) of E. coli decreased from 100% to 51.4%. It has been shown that PECVD graphene nanowalls synthesized onto G substrates, especially at 200 W, exhibited stronger antibacterial activity against E. coli and S. aureus, and the cell viabilities of E. coli and S. aureus decreased from 100% to 25.19% and 100% to 9.02%, respectively. It is concluded that that both the nanowall (3D structure) morphology, which changes significantly with the presence of RF power, and the defects created on the graphene surface using the PECVD method are more effective against E. coli and S. aureus than CVD-grown graphene-based samples (2D-structure).