Aspergillus fumigatus is an opportunistic pathogen that can cause invasive disease in immunocompromised individuals and, less frequently, in immunocompetent hosts. Proteomic investigation of A. fumigatus has the potential to enable global analysis of protein expression, identify potential targets for vaccine or diagnostic tool development, and characterise system-wide responses to external stimuli. Implementation of a large-scale proteomic strategy lead to the identification of non-redundant proteins from mycelia (n = 390) and culture supernatants (n = 42) of A. fumigatus. Utilisation of MS-based proteomics facilitated the identification of proteins typically under-represented in 2D-PAGE proteome maps, including proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Pre-fractionation of complex protein samples, by gel-filtration or gold nanoparticle pre-incubation, demonstrated potential for reduction of sample complexity. Indirect identification of secondary metabolite cluster expression was achieved using a global MS-based proteomic approach, with proteins (n = 20) from LaeA-regulated clusters detected. Targeted immunoproteomics resulted in the identification of antigenic proteins (n = 25) from A. fumigatus, reactive with sera from healthy individuals, and characterisation of these proteins may shed light on the pathobiology of A. fumigatus. Mechanisms involved in the interaction of A. fumigatus with gliotoxin were also examined, using phenotypic analysis, comparative proteomics and metabolomics. Gliotoxin was observed to relieve H2O2-induced stress, in a dose-dependent manner (0 - 10 μg/ml) and this correlated with a significant increase in expression of the gliotoxin oxidoreductase GliT (p < 0.05). This indicates a role for gliotoxin, and potentially GliT, in relief of oxidative stress in A. fumigatus. Correspondingly, proteins associated with response to stress were observed to significantly decrease in expression in the co-addition condition, relative to H2O2 alone (p < 0.05). Comparative proteomic profiling of the gliotoxin-sensitive mutant, A. fumigatus ΔgliK, revealed perturbation of translation, the methyl cycle and the endoplasmic reticulum in response to gliotoxin. This informs on the mechanisms involved in gliotoxin-mediated toxicity and may apply to other gliotoxin-sensitive species. Loss of gliotoxin production in A. fumigatus ΔgliK correlated with significant elevation in intracellular ergothioneine levels (p < 0.001). This study describes the first identification of ergothioneine in A. fumigatus and represents a target for future redox investigations.