The limitations of the therapeutic antifungals are becoming increasingly apparent in the clinic due to their modest efficacy against life-threatening systemic fungal infections. These antifungals belong to only a few structural classes that affect a small range of targets, some are quite toxic in humans while the use of others, particularly the azole drugs, has encouraged the emergence of resistant clinical isolates and the selection of innately resistant fungal pathogens. Only a few new drugs based on novel targets are in clinical development, and these may be insufficient to overcome the changing tide of fungal disease. In parallel with the successful completion of the Saccharomyces cerevisiae and human genome sequencing projects, an increasing number of genome sequencing projects are being initiated and completed for significant fungal pathogens. The growing repository of genomic information, which is complemented by decades of genetic and biochemical study, is now available for genome-wide analysis of gene function and for incisive inter-genomic comparison, with the S. cerevisiae and human genomes providing key points of reference. Functional genomic and comparative genomic techniques, many of which were developed with S. cerevisiae, are being applied to fungal pathogens with the aim of obtaining an integrated view of fungal biology and to extract targets suitable for drug discovery. This review describes some of these techniques, their limitations and their increasing contribution to the antifungal discovery process through effective gene annotation, target identification and prioritization, and in the optimization of antifungal leads.