Aspergillus fumigatus is an important fungal pathogen that represents a major threat for severely immunocompromised patients. Cases of invasive aspergillosis are associated with a high mortality rate, which reflects the limited treatment options that are currently available. The development of novel therapeutic approaches is therefore an urgent task. An interesting compound is fludioxonil, a derivative of the bacterial secondary metabolite pyrrolnitrin. Both agents possess potent antimicrobial activity against A. fumigatus and trigger a lethal activation of the group III hybrid histidine kinase TcsC, the major sensor kinase of the High Osmolarity Glycerol (HOG) pathway in A. fumigatus. In the current study, we have characterized proteins that operate downstream of TcsC and analyzed their roles in the antifungal activity of fludioxonil and in other stress situations. We found that the SskA-SakA axis of the HOG pathway and Skn7 can independently induce an increase of the internal glycerol concentration, but each of these individual responses amounts for only half of the level found in the wild type. The lethal fludioxonil-induced ballooning occurs in the sskA and the sakA mutant, but not in the skn7-deficient strain, although all three strains show comparable glycerol responses. This indicates that an elevated osmotic pressure is necessary, but not sufficient and that a second, decisive and Skn7-dependent mechanism mediates the antifungal activity. We assume that fludioxonil triggers a reorganization in the fungal cell wall that reduces its rigidity, which in combination with the elevated osmotic pressure executes the lethal expansion of the fungal cells. Two findings link Skn7 to the cell wall of A. fumigatus: (1) the fludioxonil-induced massive increase in the chitin content depends on Skn7 and (2) the skn7 mutant is more resistant to the cell wall stressor Calcofluor white. In conclusion, our data suggest that the antifungal activity of fludioxonil in A. fumigatus relies on two distinct and synergistic processes: A high internal osmotic pressure and a weakened cell wall. The involvement of Skn7 in both processes most likely accounts for its particular importance in the antifungal activity of fludioxonil.