Purine nucleotide biosynthesis and homeostasis plays a critical role in most cellular processes, including signal transduction, protein translation, DNA replication, RNA synthesis, and energy metabolism. While this extremely well conserved and characterized pathway plays a crucial role as a therapeutic target in humans, most famously for immunosuppressants and anticancer agents, it has not been well studied as a potential target for the development of antifungal drugs. Exploiting this, we have utilised a combination of genetic, biochemical and structural biology approaches to investigate the viability of purine biosynthesis as a chemotherapeutic target for the treatment of life-threatening disseminated fungal infections. Beginning with the rate-limiting enzyme required for de novo GTP synthesis, IMP dehydrogenase (IMPDH), and the key salvage enzyme required for the scavenging of guanine, hypoxanthine-xanthine-guanine phosphoribosyltransferase, we initiated a comprehensive dissection of the entire purine biosynthetic pathway in the fungal pathogen Cryptococcus neoformans. We found that de novo GTP biosynthesis, but not salvage, is critical to cryptococcal virulence in vivo in two animal models, and in contrast to other organisms, the IMPDH mutant displays a variety of phenotypic defects including slow growth and attenuated virulence factor production. With the aid of X-ray crystallography and detailed steady-state enzyme kinetics we initiated a large-scale drug screen, identifying lead compounds with the potential to serve as the basis of fungal-specific IMPDH inhibitors. As the IMPDH story continues, we have expanded our study of the de novo purine biosynthetic pathway, methodically creating deletion mutants, determining crystal structures and developing detailed kinetic profiles of additional enzymes in the pathway. Together, these results are not only supporting the potential of purine biosynthesis as a viable antifungal target, but also helping us develop a deeper understanding of this important primary metabolic pathway in the fungi.