The fight against resistance to antifungal drugs requires a better understanding of the underlying cellular mechanisms. In order to gain insight into the mechanisms leading to antifungal drug resistance, we performed a genetic screen on a model organism,  Schizosaccharomyces pombe , to identify genes whose overexpression caused resistance to antifungal drugs, including clotrimazole and terbinafine. We identified the  phb2   +  gene, encoding a highly conserved mitochondrial protein, prohibitin (Phb2), as a novel determinant of reduced susceptibility to multiple antifungal drugs. Unexpectedly, deletion of the  phb2   +  gene also exhibited antifungal drug resistance. Overexpression of the  phb2   +  gene failed to cause drug resistance when the  pap1   +  gene, encoding an oxidative stress-responsive transcription factor, was deleted. Furthermore,  pap1 +   mRNA expression was significantly increased when the  phb2   +  gene was overexpressed or deleted. Importantly, either overexpression or deletion of the  phb2   +  gene stimulated the synthesis of NO and reactive oxygen species (ROS), as measured by the cell-permeant fluorescent NO probe DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) and the ROS probe DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate), respectively. Taken together, these results suggest that Phb2 dysfunction results in reduced susceptibility to multiple antifungal drugs by increasing NO and ROS synthesis due to dysfunctional mitochondria, thereby activating the transcription factor Pap1 in fission yeast.