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Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections. Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis. Here, we exploit the diploid status of C. albicans by constructing a library of 2,868 heterozygous deletion mutants and screening this collection using 35 known or novel compounds to survey chemically induced haploinsufficiency in the pathogen. In this reverse genetic assay termed the fitness test, genes related to the mechanism of action of the probe compounds are clearly identified, supporting their functional roles and genetic interactions. In this report, chemical–genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms. We also demonstrate how chemically induced haploinsufficiency profiles can be used to identify the mechanism of action of novel antifungal agents, thereby illustrating the potential utility of this approach to antifungal drug discovery., Author Summary Candida albicans is the principal human fungal pathogen responsible for life-threatening fungal infections. Despite an urgent need for more efficacious antifungal agents, the pace of discovery has waned using the traditional approaches. In part, this reflects the longstanding limitation of performing mechanism-of-action–based screening directly in those key fungal pathogens for which new antifungal agents are sought. Here we describe an alternative approach, first developed in Saccharomyces cerevisiae and termed the fitness test, to survey approximately 45% of the C. albicans genome for the molecular targets of growth inhibitory compounds. We demonstrate that mechanistically characterized compounds can be used as chemical probes to assist gene function annotations in C. albicans. Similarly, fitness tests performed using newly discovered compounds provide powerful insights into their mechanism of action and therapeutic potential as antifungal agents. Extending this screening paradigm to C. albicans facilitates a pathogen-focused approach to antifungal drug discovery.