Fungal diseases impact the lives of millions of people across the globe. The opportunistic human fungal pathogen Cryptococcus neoformans causes cryptococcal meningitis in immunocompromised individuals with high fatality rates in response to limited treatment options. Moreover, the emergence of azole-resistant isolates in the clinic following prolonged treatment regimes, environmental fungicide exposure, and fungal evolution, threatens the outcome of current therapeutic options, endangering the survival of infected individuals. By quantitatively characterizing the proteomes of fluconazole-susceptible and -resistant C. neoformans strains using state-of-the-art tandem mass spectrometry combined with advanced bioinformatics platforms, we defined ClpX, an ATP-dependent unfoldase, as a target to overcome resistance. We discovered that disruption of ClpX through deletion or inhibition re-introduces fluconazole susceptibility into the resistant strains, rendering treatment effective once again. We further explored the mechanism of susceptibility and determined interruption to heme and ergosterol production associated with ClpX. Lastly, we are integrating in silico models of compound binding prediction to uncover novel strategies to specifically inhibit cryptococcal ClpX while limiting off-target effects against the host. Our results contribute to the understanding of novel mechanisms driving fluconazole resistance and provide support for targeting proteins as a therapeutic strategy to combat resistance.