Invasive fungal infection is usually caused by Candida albicans infection, which has a high incidence rate and mortality in critically ill patients. New drugs are needed to combat this pathogen since the limited treatment options currently available and increasing resistance to existing drugs. Berberine (BBR) is an active compound in Coptis chinensis, Phellodendron chinense and Radix berberidis, which is clinically used to treat inflammatory bowel disease, but its inhibitory effect on drug-resistant fungi has not been clarified. In this study, based on the evidence of BBR inhibiting the expression of azole-resistance genes, reducing cell adhesion and disrupting biofilm formation, transcriptome analysis revealed that the disruption of iron acquisition pathway may be the core link in BBR inhibiting drug-resistant fungi. Combined with the subsequent experimental results, including the reduction of intracellular ferrous ion content, the weakening of iron reductase activity and the overall downregulation of the coding gene of the high-affinity iron reduction system, it is speculated that the fungal growth defect under BBR treatment is the result of the interruption of the high-affinity iron acquisition pathway. Ftr1 plays a central role in the drug targeting of this transport system. Meanwhile, due to the iron deficiency within the cell, the biological function of mitochondria is impaired, ultimately leading to fungal death. This study not only reflects the application value of BBR in the clinical treatment of fungal infections, but also provides a potential strategy to address the current drug-resistance dilemma.