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UNLABELLED: (formerly ) is a leading cause of invasive candidiasis and rapidly develops antifungal drug resistance during treatment. An increasing number of clinical isolates show reduced susceptibility to echinocandins and azoles, leaving amphotericin B (AMB) as a last therapeutic option. Resistance of to this drug is rare, and its underlying mechanisms are still not fully understood. Here, we describe two independent multidrug-resistant bloodstream isolates displaying resistance to AMB and anidulafungin (ANF), as well as a reduced susceptibility to azoles. We performed whole-genome sequencing and sterol profiling on nine clinical isolates, which were resistant to ANF and displayed resistance or low susceptibility to fluconazole (FLU) and AMB. We identified loss-of-function mutations in the genes and , which could be linked to ergosterol depletion and AMB resistance. The transcriptional response of the reference strain CBS138 and an AMB + ANF isolate was analyzed by RNA-seq, revealing that ergosterol depletion also contributed to upregulation of and ABC transporter genes, which might explain the low FLU susceptibility. Surprisingly, the AMB isolates displayed severe fitness defects, and one of them was fully virulent in a infection model. Our results indicate that ergosterol depletion in leads to AMB resistance without affecting fitness or virulence.IMPORTANCE: The major human fungal pathogen is well known for its fast development of antifungal drug resistance, especially against commonly used azoles. However, it can also acquire resistance to echinocandins, leading to multidrug resistance (MDR) and leaving amphotericin B (AMB) as the last therapeutic option. AMB resistance is rare, mainly caused by ergosterol depletion, and is normally associated with severe fitness costs for the pathogen. However, we found bloodstream isolates with stable AMB resistance without apparent fitness and virulence defects. The underlying ergosterol depletion contributed to low azole susceptibility and was associated with anidulafungin resistance. These findings demonstrate how fast MDR can evolve in and underline the need for close resistance monitoring.