Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic and allergic Aspergillus infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets however, development of similar systems in filamentous fungi are complicated by the fact that they undergo cell fusion and heterokaryosis. Here we demonstrate that cell fusion in A. fumigatus under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors. Using this knowledge, we have developed a barcode-free genetic profiling system that permits high throughput parallel determination of strain fitness in a collection of diploid A. fumigatus mutants. We show heterozygous null mutants in cyp51A and arf2 have reduced fitness in the presence of Itraconazole and Brefeldin A respectively and a heterozygous null of atp17 is resistant to Brefeldin A.