Ergothioneine (EGT) is a tri-N-methylated and sulphurised histidine derivative which exhibits antioxidant properties. EGT was first identified in Aspergillus fumigatus, in work pertaining to gliotoxin biosynthesis. Compared to wild-type, EGT levels were elevated in the absence of GliK, a gliotoxin biosynthetic enzyme. The work presented in this thesis demonstrates that deletion of A. fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. A second EGT biosynthetic enzyme, egtB (AFUA_2G13295), was also identified. EgtB, a pyridoxal phosphate (PLP)-dependant cysteine desulphurase, contributed to, but was not essential for, EGT biosynthesis. EGT absence in A. fumigatus ΔegtA significantly reduced resistance to elevated concentrations of H2O2 and menadione, impaired gliotoxin production, increased glutathione biosynthesis, resulted in attenuated conidiation and affected cell wall integrity. EGT deficiency specifically decreased resistance to high H2O2 levels, which strongly infers functionality as an auxiliary, high-level, antioxidant. Quantitative proteomic analysis revealed significant proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to affect EGT biosynthesis. Quantitative proteomic analysis also identified VipC (AFUA_8G01930), a putative methyltransferase which may be involved in the A. fumigatus response to EGT absence. A vipC deletion mutant was thus generated to further investigate VipC function in A. fumigatus. ΔvipC demonstrated significantly altered growth, development and metabolism compared to wild-type. Glutathione levels and abundance of related biosynthetic enzymes were significantly decreased in ΔvipC. Additionally, enzymes involved in the methionine cycle also had decreased abundance. These data indicate that VipC plays a significant role in coordinating the metabolic response to EGT absence in A. fumigatus. As a sulphur-containing metabolite, EGT therefore plays a key role in redox homeostasis and requires essential consideration in future oxidative stress studies in A. fumigatus.