Strong antifungal efficacy against C. neoformans and to understand the mechanism of action of the compound. We used C. neoformans because of its clinical importance, well-annotated genome sequence, and robust genetic tools. A series of vanillin derivatives, including hydroxy and alkoxy benzaldehydes, halogenated benzaldehydes, and nitrated benzaldehydes, were tested for their antifungal activity against C. neoformans. We found a structural correlation between the vanillin derivatives and antifungal activity against C. neoformans; that is, the hydroxyl or alkoxy group appeared to be more advantageous than the halogenated or nitrated group in benzaldehyde. Among the vanillin derivatives with a hydroxyl or alkoxy group, o-vanillin and o-ethyl vanillin showed the highest antifungal activities against C. neoformans. We chose o-vanillin to study the mechanism of action. To date, numerous studies have successfully used functional genomics approaches, in particular transcriptome analysis, to identify the target pathway of currently available antifungal drugs and novel antifungal AZD6244 candidate drugs. We therefore used transcriptome analysis to understand the mechanism of action of o-vanillin. Global transcript profiles of C. neoformans cells treated with ovanillin were obtained using RNA sequencing and were compared with the transcript profiles of the same strain not treated with the compound. The results of our transcriptome analysis suggested that o-vanillin likely acts by significantly reducing mitochondrial function, which would in turn disrupt cellular redox-homeostasis in C. neoformans. Our hypothesis was experimentally confirmed by the observation of hypersensitivity of the C. neoformans mutants lacking the genes involved in the oxidative stress response upon treatment with o-vanillin. We further investigated depletion of mitochondrial function by o-vanillin using dihydrorhodamine 123, which is a fluorescent dye that stains mitochondria in living cells. The damaged mitochondria retained much less dihydrorhodamine 123 than did the healthy mitochondria. We treated the fungal cells with o-vanillin and dihydrorhodamine 123 and analyzed the fluorescence intensity by fluorescence-activated cell sorting. In untreated cells, suggesting that mitochondrial functions were damaged by the compound. Our results were also supported by the observation of less accumulation of dihydrorhodamine 123 in the cfo1 mutant and the ccp1 mutant, which displayed significantly reduced mitochondrial function. Taken together, the results of the present study suggest that ovanillin has antifungal activity against C. neoformans and substantially distorts mitochondrial functions, resulting in an overall deficiency of oxidative stress defense mechanisms in C. neoformans. Therefore, o-vanillin can be an effective drug candidate to treat cryptococcosis.