Mycetoma is a chronic granulomatous infection of the subcutaneous tissue, most often caused by the fungal pathogen Madurella mycetomatis. Characteristic of the infection is the formation of grains. However, knowledge of the function and formation of the grain is limited. To map the processes leading to M. mycetomatis grain formation, we used a Galleria mellonella larvae infection model and transcriptomic profiling. G. mellonella larvae were infected with M. mycetomatis and after 4h, 24h, 72h and 168h post-inoculation, RNA was extracted from larval content and sequenced. Differential Gene Expression analysis revealed that 3,498 G. mellonella and 136 M. mycetomatis genes were differentially expressed during infection. Among these genes, genes related to iron transport were highly expressed by both G. mellonella (transferrin and ferritin) and M. mycetomatis (SidA, SidD and SidI). LC-MS/MS analysis of M. mycetomatis cultured under iron-limiting conditions revealed the presence of SidA and SidD orthologs and concurrent RP-HPLC and LC-MS identified a singly charged, putative siderophore in culture supernatant. M. mycetomatis could also obtain iron from holoferritin. The identification of the importance of iron acquisition during grain formation can be exploited as a potential novel diagnostic and therapeutic strategy for mycetoma.