Herein, we describe interfacially-assembled [7]helicene films that were deposited on graphene monolayer using the Langmuir-Schaefer deposition by utilizing the interactions of nonplanar (helicene) and planar (graphene) π–π interactions as functional antifouling coatings. Bacterial adhesion of Staphylococcus aureus on helicene—graphene films was noticeably lower than that on bare graphene, up to 96.8% reductions in bacterial adhesion. The promising bacterial antifouling characteristics of helicene films was attributed to the unique molecular geometry of helicene, i.e., nano-helix, which can hinder the nanoscale bacterial docking processes on a surface. We envision that helicene—graphene films may eventually be used as protective coatings against bacterial antifouling on the electronic components of clinical and biomedical devices.