Alveolar bone loss is usually treated with guided bone regeneration, a dental procedure which utilizes a tissue-separation membrane. The barrier membrane prevents pathogens and epithelial cells to invade the bone augmentation site, thereby permitting osteoblasts to deposit minerals and build up bone. This study aims at adding bioactive properties to otherwise inert PTFE membranes in order to enhance cell adherence and promote proliferation. A prewetting by ethanol and stepwise hydration protocol was herein employed to overcome high surface tension of PTFE membranes and allow for a recombinant spider silk protein, functionalized with a cell-binding motif from fibronectin (FN-silk), to self-assemble into a nanofibrillar coating. HaCaT and U-2 OS cells were seeded onto soft and hard tissue sides, respectively, of membranes coated with FN-silk. The cells could firmly adhere as early as 1 h post seeding, as well as markedly grow in numbers when kept in culture for 7 days. Fluorescence and scanning electron microscopy images revealed that adherent cells could form a confluent monolayer and develop essential cell-cell contacts during 1 week of culture. Hence, functionalized PTFE membranes have a potential of better integration at the implantation site, with reduced risk of membrane displacement as well as exposure to oral pathogens.