The non-ribosomal peptide gliotoxin, is a toxic fungal secondary metabolite produced by the filamentous fungus Aspergillus fumigatus. Previous work has implicated its potent anti-fungal properties and importance as a virulence factor in human infection. Availability of the A. fumigatus genome has allowed for the characterisation of the gliotoxin biosynthetic cluster gli, with previous work illustrating the importance of the thioredoxin reductase gliT in self protection against gliotoxin, regulation of which has been shown to be independent of the transcriptional role of gliZ in regulating the cluster. The work presented here characterises the creation of a double gene deletion strain lacking both gliT and gliZ (ΔgliTΔgliZ) ultimately silencing the gli cluster. Phenotypic characterisation revealed that ΔgliTΔgliZ is more sensitive to gliotoxin challenge when compared with ΔgliZ, yet is more resistant when compared with ΔgliT, highlighting the importance of gliT in protection against gliotoxin, especially when the cluster is still expressed. An anti-oxidant role for gliotoxin is highlighted, with co-addition of gliotoxin and hydrogen peroxide resulting in a reduction in the production of reactive oxygen species in A. fumigatus, when compared with hydrogen peroxide only treated cells. Additionally, proteomic and transcriptomic data indicate gliotoxin exposure dysregulates S-adenosyl-L-methionine biosynthesis with key enzymes of this pathway, i.e., S-adenosyl-L-homocysteinase and cobalmin-independent L-methionine synthase, eliciting significant changes in their respective expression in response to gliotoxin in A. fumigatus an also in Saccharomyces cerevisiae. Using a gene candidate approach, S. cerevisiae Δsod1 and Δyap1 sensitivity to exogenous gliotoxin suggest gliotoxin induces some form of oxidative stress on the cell. Furthermore, low to null levels of glutathione were seen to be advantageous to the S. cerevisiae mutant strain Δgsh1, eliciting resistance to gliotoxin challenge. Similarly, glutathione levels were found to significantly altered in A. fumigatus gliT and gliZ mutant lysates and may be a factor in their respective gliotoxin sensitivity. Overall this work highlights key factors which may contribute to gliotoxin toxicity. Highlighting the importance of gliotoxin biosynthetic genes and the pathways involved in the response to gliotoxin exposure.