It is estimated that over 150 million individuals suffer from serious fungal infections, resulting in 1.5 million deaths a year. Aspergillus fumigatus is a filamentous saprophytic fungus. However, in hosts with an altered immune system it can cause a variety of diseases. The most serious is invasive aspergillosis, which is estimated to cause over 200,000 life-threatening infections annually with high mortality rates. Fungal diseases are increasing due to the expansion of the immunocompromised cohorts of patients. The current antifungal arsenal is limited and paired with severe sides effects. Additionally, antifungal resistance is on the rise. To develop new antifungals, a mechanistic understanding of A. fumigatus pathogenicity is required. This thesis will address current methodologies available to phenotype large collections of mutants for fitness, infection-related stresses and invasion and will identify transcription factors required for these processes by utilising the genome-wide transcription factor knockout library. Gene expression is tightly regulated at the transcriptional level. This project outlines the importance of transcription factors in azole resistance as a pilot phenotype screening and explores the regulatory mechanism of the multi-drug resistant negative cofactor transcription factors, NctA and NctB. These key regulators control many processes, including ergosterol biosynthesis, a direct target of the azoles. Additionally, the transcription factor knockout library is screened under infection-related stresses, for detachment and cytotoxicity of epithelial cells. This first in field screening identifies previous uncharacterised transcription factors and explores new phenotypes for previous characterised transcription factors. Furthermore, regulators required for environmental adaptation are a distinct set from ones required for epithelial invasion. This provides the first evidence for the accidental pathogen hypothesis. Lastly, the genome editing technique CRISPR-Cas9 is explored to allow marker-free transformation in A. fumigatus. This technique can be used for gene replacement and epitope tagging without the need of labour- and time-intensive construct generation. Furthermore, marker-free transformation will reduce the chance of off-target effects.