Pathogenic microbial contaminations on the surface of e.g. medical products and the associated risk of infection are a severe problem especially in the public health care sector. Furthermore, antimicrobial resistant strains are increasing at an alarming rate due to the overuse of antimicrobial agents. Since one critical step in biofilm formation is the initial adherence of pathogenic microbes onto a material’s surface, inhibiting microbial attachment is a reasonable approach to develop material surfaces resistant to biofilm formation. There are two main strategies for inhibiting surface attachment, referred to as either active or passive resistance. While passively resistant surfaces utilize super hydrophilic or hydrophobic polymers, zwitterionic and other synthetic polymers, actively resistant ones include contact killing materials such as cationic polymers, amphiphilic polymers, antimicrobial peptides and polymeric/ composite materials loaded with antimicrobial agents. In this work, a novel passive approach was developed originating from the basic observation that some silk materials display high resistance against microbial degradation. A new material platform of different recombinantly produced spider silk proteins based on the consensus sequences of Araneus diadematus dragline silk proteins (fibroin 3 and 4) was applied to produce 2D-patterned films and 3D-hydrogels. The here processed biotechnologically designed materials feature varying structural characteristics on the nanoscale resulting in differently pronounced anti-fouling properties when exposed to pathogenic bacteria (S. mutans, S. aureus, E. coli) and fungi (C. albicans, P. pastoris). Strikingly, recombinant spider silk proteins could be designed in a bio-selective manner that promoted mammalian cell attachment and proliferation (BALB/3T3 fibroblasts) while simultaneously inhibiting microbial infestation without the need of further additives. This intrinsic bio-selective behavior is suspected to base on crystal size and distribution resulting in microbe-repellent self-assembled nano-topographies, whereas functionalization with cell-binding motifs simultaneously promotes mammalian cell adhesion. The findings indicate the great potential of engineered spider silk-based materials as anti-fouling coatings in biomedical and technical applications as well as for hydrogel-based tissue regeneration.