ABSTRACTThermotolerance is a remarkable virulence attribute ofAspergillus fumigatus, but the consequences of heat shock (HS) to the cell membrane of this fungus are unknown, although this structure is one of the first to detect changes in ambient temperature that imposes on the cell a prompt adaptative response. Under high-temperature stress, fungi trigger the HS response controlled by heat shock transcription factors, such as HsfA, which regulates the expression of heat shock proteins. In yeast, smaller amounts of phospholipids with unsaturated fatty acid (FA) chains are synthesized in response to HS, directly affecting plasma membrane composition. The addition of double bonds in saturated FA is catalyzed by Δ9-fatty acid desaturases, whose expression is temperature-modulated. However, the relationship between HS and saturated/unsaturated FA balance in membrane lipids ofA. fumigatusin response to HS has not been investigated. Here, we found that HsfA responds to plasma membrane stress and has a role in sphingolipid and phospholipid unsaturated biosynthesis. In addition, we studied theA. fumigatusΔ9-fatty acid desaturasesdeAand discovered that this gene is essential and required for unsaturated FA biosynthesis, although it did not directly affect the total levels of phospholipids and sphingolipids.sdeAdepletion significantly sensitizes matureA. fumigatusbiofilms to caspofungin. Also, we demonstrate thathsfAcontrolssdeAexpression, while SdeA and Hsp90 physically interact. Our results suggest that HsfA is required for the adaptation of the fungal plasma membrane to HS and point out a sharp relationship between thermotolerance and FA metabolism inA. fumigatus.IMPORTANCEAspergillus fumigatuscauses invasive pulmonary aspergillosis, a life-threatening infection accounting for high mortality rates in immunocompromised patients. The ability of this organism to grow at elevated temperatures is long recognized as an essential attribute for this mold to cause disease.A. fumigatusresponds to heat stress by activating heat shock transcription factors and chaperones to orchestrate cellular responses that protect the fungus against damage caused by heat. Concomitantly, the cell membrane must adapt to heat and maintain physical and chemical properties such as the balance between saturated/unsaturated fatty acids. However, howA. fumigatusconnects these two physiological responses is unclear. Here, we explain that HsfA affects the synthesis of complex membrane lipids such as phospholipids and sphingolipids and controls the enzyme SdeA, which produces monounsaturated fatty acids, raw material for membrane lipids. These findings suggest that forced dysregulation of saturated/unsaturated fatty acid balance might represent novel strategies for antifungal therapy.