The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. Sequencing of the fungal genome has led to an increased understanding of the organism; however the functions of many genes remain unknown. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione s-transferase activity, in A. fumigatus. The work presented here demonstrates the role of ElfA in protein synthesis, but also that it plays a key role in the oxidative stress response and may be involved in actin cytoskeleton organisation. Phenotypic analysis demonstrates that an elfA deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H2O2 (> 1 mM) (p = 0.0003), diamide (> 0.5 mM) (p < 0.001), and 4, 4’-dithiodipyridine (> 7.5 μM) (p = 0.0056), and was significantly more resistant to voriconazole (> 0.5 μg/ml) (p = 0.0251) than the wild-type. Comparative proteomics, under basal and oxidative stress conditions, revealed significantly (p < 0.05) altered expression of proteins involved in protein synthesis, the oxidative stress response and actin cytoskeleton organization. In parallel, comparative proteomics of A. fumigatus Δpes3, a non-ribosomal peptide synthetase deficient strain, supports a structural role for this peptide with the altered expression of proteins functioning in morphogenesis and germination. A. fumigatus Δpes3 exhibited a reduced germination rate in static cultures and confocal microscopy revealed not only shorter germ tubes but reduced surface (1,3)-β-glucan in this mutant. Moreover, the reduced surface (1,3)-β-glucan explains the lower MIC of A. fumigatus Δpes3 to voriconazole measured in this study as surface (1,3)-β-glucan sequesters azoles preventing entry into the cell. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to protein synthesis, oxidative stress and actin cytoskeleton organisation. In addition to this, the advantages of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function are also revealed.