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Every year approximately 1.5 million people die due to systemic fungal infections. Issues with diagnosis, limited treatment options and resistance to antifungal drugs have led to a high mortality rate. Currently, only five classes of antifungal drugs are available: the azoles, the polyenes (amphotericin B), pyrimidine analogues (flucytosine), the echinocandins and the allylamines/thiocarbamates. The azoles are frequently the first line of treatment, but the emergence of resistance against drugs in this class is a growing problem. Although new drugs are approved for clinical use occasionally, they usually belong to an existing class. The development of new antifungal drugs, acting on new targets is difficult, due to the phylogenetic relatedness between human and fungi: potential antifungal drugs often have unacceptably high levels of toxicity, making them unsuitable for treatment. Recently, F901318, a first-in-class orotomide antifungal developed by F2G Ltd., has gone into clinical development for invasive aspergillosis. During this PhD project, the effects of F901318 on the human pathogenic fungus Aspergillus fumigatus were investigated. F901318 inhibits dihydroorotate dehydrogenase (DHODH), thereby blocking the de novo pyrimidine biosynthesis pathway. Pyrimidines play key roles in several important cellular processes, such as DNA/RNA synthesis, cell cycle regulation, protein synthesis and cell wall synthesis. Live-cell, time lapse confocal fluorescence microscopy was utilized to analyze the effect of DHODH inhibition by F901318 in A. fumigatus. Growth and viability was analyzed, as well as the morphology of intracellular structures affected by pyrimidine biosynthesis inhibition. Furthermore, a fluorescent analogue of F901318 was demonstrated to be a valuable tool in analyzing drug uptake and distribution. Together, the results have led to the conclusion that F901318 is a time-dependent fungicidal drug, by causing growth arrest and hyphal swelling that over time leads to cell lysis. Morphological rearrangements of multiple cellular structures, such as the cell wall, the vacuoles and the nuclei, show that inhibition of the pyrimidine biosynthesis pathway affects many key cellular processes.