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The rising rate of drug-resistant fungal infections and the emergence of intrinsically resistant pathogens pose growing clinical challenges. Because fungi are closely related to mammals, developing antifungals without toxic off-target effects is difficult. Targeted gene repression can model drug-mediated inhibition and reveal gene dosage sensitivity, but traditional approaches in the fungal pathogen Candida albicans are labour intensive and low throughput. Here we adapt pooled CRISPR interference (CRISPRi) screening in C. albicans to enable large-scale functional genomic analysis. We assess repression sensitivity of 130 essential genes conserved in fungi without close homologues in humans and identify highly dosage-sensitive genes across multiple pathways. Screening across ten environmental conditions reveals environment-dependent effects on gene sensitivity. Extending these experiments to two drug-resistant clinical isolates shows that many fitness defects are conserved across genetic backgrounds. Thus, CRISPRi pooled screening enables rapid, large-scale functional genomics across diverse genetic backgrounds in C. albicans.