Biofouling, the unwanted adhesion of organisms to surfaces, has a negative impact on energy, food, water, and health resources. One possible strategy to fight biofouling is to modify the surface using a peptide-based coating that will change the surface properties. We reveal the importance of rational design and positioning of individual amino acids in an amphiphilic peptide sequence. By just manipulating the position of the amino acids within the peptide chain having the same chemical composition, we improved the antifouling performance of an amphiphilic peptide-based coating, Phe(4-F)-Lys-DOPA, by 30%. We have judiciously tailored the peptide configurations to achieve the best antifouling performance by (i) positioning the amino acid lysine adjacent to the DOPA moiety in the linear peptide chain for better adhesion, (ii) having a linear fluorinated N-terminal to improve the packing density of the film by straightening the peptide chain, and (iii) placing DOPA at the C-terminal. We have also compared the antifouling performances of amphiphilic, hydrophobic, hydrophilic, and alternately arranged peptides. Our results show a reduction of ∼80% in bacterial adhesion for an amphiphilic peptide-coated surface when compared to a bare titanium surface. This work provides important strategic design guidelines for future peptide-related materials that have effective antifouling properties.