The polyene antifungal amphotericin B (AmB) can form giant helical aggregates. We show that microscopic and mesoscopic structural features of its aggregates can be revealed by Raman optical activity (ROA) and electronic circular dichroism (ECD), respectively. The ROA method, which senses molecular structure more locally, elucidates the conformation of the polyene chain of individual AmB molecules in an aggregated state. In turn, the ECD signal related to exciton coupling effects provides details about the arrangement of AmB molecules in the supramolecular structure. Thus, the use of both complementary methods will be crucial in future structural studies of chiral supramolecular systems.