Amphotericin B (AMB) is a classical polyene antifungal agent with powerful and broad-spectrum activity against pathogenic fungi, and less likely to be resistant. Aspergillus terreus, a clinically common pathogenic Aspergillus, is gaining attention due to intrinsic AMB resistance. At present, the mode of antifungal action of AMB needs to be further elucidated, and little is known about the mechanism of AMB resistance. This review addresses the novel mode of action by which AMB induces increased endogenous reactive oxygen species (ROS) production in Aspergillus terreus. Also, the role of stress adaptation pathways (heat shock protein 90, heat shock protein 70, and calcineurin), ROS scavenging enzymes, as well as mitochondrial function in the mechanisms of AMB resistance in Aspergillus terreus is highlighted. These novel findings are of great significance in understanding both action and resistance mechanism of AMB in other pathogenic Aspergilli spp. with clinical importance.