Amphotericin B (AmB) is a lifesaving antibiotic used to treat deep-seated mycotic infections. Both the pharmaceutical activity and highly toxic side effects of the drug rely on its interaction with biomembranes, which is governed by the molecular organization of AmB. In the present work, we present a detailed analysis of self-assembly of AmB molecules in different environments, interesting from the physiological standpoint, based on molecular spectroscopy techniques: electronic absorption, circular dichroism, steady state and time-resolved fluorescence and molecular dynamic calculations. The results show that, in the water medium, AmB self-associates to dimeric structures, referred to as "parallel" and "antiparallel". AmB dimers can further assemble into tetramers which can play a role of transmembrane ion channels, affecting electrophysiological homeostasis of a living cell. Understanding structural determinants of self-assembly of AmB opens a way to engineering preparations of the drug which retain pharmaceutical effectiveness under reduced toxicity.