Antibacterial activity and cytocompatibility are the two essential characteristics that an ideal implantable biomaterial must possess simultaneously. Biomaterials with these characteristics can be fabricated via combination of chemical and topographical features. Herein, design and fabrication of a sharkskin mimicked Graphene Oxide modified Chitosan membrane with enhanced antibacterial and cytocompatibility properties was investigated. As a measure of antibacterial properties, viability of planktonic and bacterial biofilm was measured using gram-positive Staphylococcus aureus and gram-negative Escherichia coil. Results showed a significant reduction in bacterial adhesion and biofilm growth induced by sharkskin surface topography regardless of chemical modifications for both strains, hence proving the superior antibacterial activity of sharkskin topography. Furthermore, the highest level of cell viability and proliferation of cultured human keratinocyte (HaCaT) and mouse fibroblast (L929) cell lines belonged to Graphene Oxide (GO) coated sharkskin mimicked membranes. Our results indicate that GO coated (GOc) sharkskin mimicked membranes can significantly reduce bacterial biofilm formation in stationary culture conditions while promoting cytocompatibility. The duo of sharkskin surface topography and GO coating provides remarkable potentials as a cytocompatible and antibacterial biomaterial for diverse biomedical applications.