Bacterial accumulation on solid material displays a major source of biomaterial associated infections, cross contamination, and spreading. To overcome these problems, different investigations on surface modifications for the containment of bacterial adhesion have been done. The aim of this research is the development of a rapid and efficient screening procedure to identify and investigate biologically active peptides in an immobilized state in order to produce an antimicrobial surface coating. We figured out that the antimicrobial mode of action is the most important parameter because only peptides with pronounced membrane disruption abilities displayed meaningful activity in an immobilized state. In addition, we highlighted the influence of the coupling reaction chemistry on the activity and amount of the immobilized peptide. Thereupon we developed an optimized antimicrobial surface coating with unrestricted antimicrobial properties by adjusting the immobilization strategy in combination with lowering the necessary peptide amount. Moreover we demonstrated that this antimicrobial surface coating displayed no cytotoxic activity against a eukaryotic cell line and thereby indicates a promising biocompatibility. Furthermore, different antimicrobial peptides obtained either by chemical peptide synthesis or by recombinant DNA technology were used in this study and their activities as well as their potential applications were discussed.