Amphotericin B (AmB) is a well-known antifungal antibiotic that has been used in the clinic for about five decades. Despite its chemotherapeutic importance, AmB is quite toxic and many efforts have been made to improve its pharmacological properties, e.g., by chemical modifications. The lipid membrane is a molecular target for AmB, however, due to heterogeneity of its components, the molecular mechanism of AmB action is still unclear. The lack of this knowledge hinders rational designing of new and less toxic AmB derivatives. Our review is a critical presentation of the current understanding of AmB molecular mechanism of action at the membrane level. Except the experimental approach, the extensive overview of molecular modeling studies, performed mostly in our lab, is presented. The results of interactions between AmB or some of its derivatives and lipid model membranes are discussed. In our studies, different biomembrane models and different associate states of the antibiotic were included. Presented molecular modeling approach is especially valuable with regard to a new paradigm of the structure of lipid membrane containing liquid-ordered domains. Hopefully, all these complementary experimental/computational approaches are going to reach the point at which a new hypothesis about molecular mechanism of AmB activity and selectivity will be put forward.