Knowing the full set of essential genes for a given organism provides important information about ways to promote, and to limit, its growth and survival. For many non-model organisms, the lack of a stable haploid state and low transformation efficiencies impede the use of conventional approaches to generate a genome-wide comprehensive set of mutant strains and the identification of the genes essential for growth. Here we report on the isolation and utilization of a highly stable haploid derivative of the human pathogenic fungus Candida albicans, together with a modified heterologous transposon and machine learning (ML) analysis method, to predict the degree to which all of the open reading frames are required for growth under standard laboratory conditions. We identified 1,610 C. albicans essential genes, including 1,195 with high “essentiality confidence” scores, thereby increasing the number of essential genes (currently 66 in the Candida Genome Database) by >20-fold and providing an unbiased approach to determine the degree of confidence in the determination of essentiality. Among the genes essential in C. albicans were 602 genes also essential in the model budding and fission yeasts analyzed by both deletion and transposon mutagenesis. We also identified essential genes conserved among the four major human pathogens C. albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Histoplasma capsulatum and highlight those that lack homologs in humans and that thus could serve as potential targets for the design of antifungal therapies.