Gliotoxin (GT) is the prototype of the epidithiodioxopiperazine (ETP)-type fungal toxins. GT plays a critical role in the pathobiology of Aspergillus fumigatus. It modulates the immune response and induces apoptosis in different cell types. The toxicity has been attributed to the unusual intramolecular disulfide bridge, which is the functional motif of all ETPs. Because of the extraordinary structure and activity of GT, this fungal metabolite has been the subject of many investigations. The biosynthesis of GT involves unprecedented reactions catalysed by recently discovered enzymes. Here, we summarize the recent progress in elucidating the GT biosynthetic pathway and its role in virulence.