Vibrational circular dichroism (VCD) spectroscopy has become an excellent tool to study biological nanostructures and biomimetic materials in their functional environment and is thus complementary to otherwise employed diffraction, imaging, and spectroscopy methods. However, it is still difficult to relate the observed exceptionally large VCD signals to specific structural elements. Here, we systematically studied the VCD signatures of structurally well-defined and thoroughly characterized nanofibrils from oligopeptide-substituted perylene bisimides that comprise single parallel β-sheets. These nanofibrils show a giant VCD signal in the absence of β-sheet stacking and a negative VCD couplet despite their right-handed helicity. The giant VCD signal was very sensitive to subtle changes in the molecular structure as well as 13 C-labeling, which caused a strong disruption of the exciton system as confirmed by two-dimensional infrared spectroscopy. Simulations based on the commonly applied transition dipole coupling model qualitatively reproduced the IR spectra but failed to account for the observed giant VCD or the strong isotope effect. Because our model system and isotope labeling imposes stringent structural constraints of the observed spectroscopic features, our results challenge current assumptions regarding the structural parameters determining VCD sign and intensity. The investigated system may, hence, serve as a benchmark for more sophisticated models with better predictive power for the investigation of protein aggregates in biomedical context or novel oligopeptide-based nanomaterials.