Biofouling represents great challenges in many applications, and zwitterionic peptides have been a promising candidate due to their biocompatibility and excellent antifouling performance. Inspired by lubricin, we designed a loop-like zwitterionic peptide and investigated the effect of conformation (linear or loop) on the antifouling properties using a combination of surface plasma resonance (SPR), surface force apparatus (SFA), and all atomistic molecular dynamics (MD) simulation techniques. Our results demonstrate that the loop-like zwitterionic peptides perform better in resisting the adsorption of proteins and bacteria. SFA measurements show that the loop-like peptides reduce the adhesion between the modified surface and the modeling foulant lysozyme. All atomistic MD simulations reveal that the loop-like zwitterionic peptides are more rigid than the linear-like zwitterionic peptides and avoid the penetration of the terminus into the foulants, which lower the interaction between the zwitterionic peptides and foulants. Besides, the loop-like zwitterionic peptides avoid the aggregation of the chains and bind more water, improving the hydrophilicity and antifouling performance. Altogether, this study provides a more comprehensive understanding of the conformation effect of zwitterionic peptides on their antifouling properties, which may contribute to designing novel antifouling materials in various biomedical applications.