Noncovalent and electrostatic interactions facilitate the formation of complex networks through molecular self-assembly in biomolecules such as proteins and glycosaminoglycans. Self-assembling peptide amphiphiles (PA) are a group of molecules that can form nanofibrous structures and may contain bioactive epitopes to interact specifically with target molecules. Here, we report the presentation of cationic peptide sequences on supramolecular nanofibers formed by self-assembling peptide amphiphiles for cooperative enhanced antibacterial activity. Antibacterial properties of self-assembled peptide nanofibers were significantly higher than soluble peptide molecules with identical amino acid sequences, suggesting that the tandem presentation of bioactive epitopes is important for designing new materials for bactericidal activity. In addition, bacteria were observed to accumulate more rapidly on peptide nanofibers compared to soluble peptides, which may further enhance antibacterial activity by increasing the number of peptide molecules interacting with the bacterial membrane. The cationic peptide amphiphile nanofibers were observed to attach to bacterial membranes and disrupt their integrity. These results demonstrate that short cationic peptides show a significant improvement in antibacterial activity when presented in the nanofiber form.