Due to the narrow activity spectrum of echinocandin antifungal drugs and the emergence of drug resistance, new therapies are needed to potentiate echinocandins such as caspofungin (CAS). We have identified a sesquiterpene quinone compound named puupehenone (PUUP) that enhances CAS activity in CAS-resistant strains of Candida albicans and also in the inherently CAS-insensitive pathogen Cryptococcus neoformans. To investigate PUUP’s CAS-potentiating mechanism, we conducted a transcript profiling study in the model yeast Saccharomyces cerevisiae. Cells were exposed to solvent, PUUP, CAS, and PUUP + CAS, and the RNA extracted was subjected to RNA-Seq analysis. To further investigate PUUP’s mechanism of action, a fitness profiling study was performed to monitor the sensitivity of whole-genome yeast deletion mutants to PUUP. To confirm PUUP’s effect on Hsp90, a well-established promoter-reporter assay system was used that monitors the rat glucocorticoid receptor (GR), an Hsp90 client protein. An additional client protein, Mpk1 was monitored by Western analysis. Transcript profiling with CAS + PUUP revealed that genes in the cell wall integrity pathway (CWIP) that were strongly induced by CAS alone were not induced by CAS + PUUP; thus, PUUP synergizes with CAS by preventing cell wall repair through the CWIP. Further studies revealed that PUUP targets Hsp90. The transcript profile of PUUP was similar to that of the Hsp90 inhibitor celastrol. Genes encoding chaperones and co-chaperones involved in heat shock response were strongly induced by PUUP. To confirm that PUUP targets Hsp90, we monitored the rat GR, an Hsp90 client protein, and observed that PUUP inhibited GR induction in a concentration-dependent manner. Our fitness profiling experiment identified 28 mutants with increased hypersensitivity to PUUP. Of these, 14 mutants have been previously reported to show hypersensitivity to other Hsp90 inhibitors. We also showed that the overexpression of four different Hsp90-related proteins resulted in significant resistance to PUUP. We also observed that PUUP inhibited the activation of the yeast protein Mpk1, another Hsp90 client protein. Because Mpk1 is a critical kinase in the CWIP pathway, this result demonstrates that PUUP inhibits the CWIP by disrupting Mpk1 activity. In summary, we have identified a natural product that disrupts the fungal cell wall integrity pathway by targeting Hsp90.