Triazoles are the mainstay of treatment for aspergillosis, although resistance to these antifungal agents may be associated with treatment failure. Refractory infections often necessitate a switch to other antifungal agents, including amphotericin B (AmB), although these infections may not resolve. The aim of this study was to investigate the effect of prior azole exposure on AmB sensitivity in Aspergillus fumigatus biofilms. It was hypothesised that sequential antifungal therapy has the potential to impact adaptive resistance mechanisms. Antifungal sensitivity was determined for each isolate against AmB±voriconazole (VRZ) exposure by a broth microdilution method and an XTT metabolic assay. To analyse the role of extracellular DNA (eDNA) and Hsp90 activation, sensitivity to AmB±DNA-digesting enzyme (DNase) or Hsp90 inhibitor [geldanamycin (GDA)] was also tested. Finally, scanning electron microscopy was performed to assess phenotypic changes. The in vitro data revealed that A. fumigatus sensitivity to AmB was decreased when it was tested in combination with VRZ. In addition, a two- to four-fold decreased sensitivity to AmB was recorded against VRZ-exposed germlings compared with controls. It was also shown that depletion of eDNA by DNase treatment enhanced AmB activity against VRZ-exposed cells by eight-fold, which visually could be explained by destabilisation of the biofilm when examined microscopically. Pharmacological inhibition of Hsp90 by GDA significantly improved biofilm susceptibility to AmB by four- to eight-fold. In conclusion, A. fumigatus pre-exposure to VRZ concomitantly induces eDNA release and activates the stress response, which collectively confers AmB resistance in vitro.