Abstract： Objective To explore the susceptibility of Aspergillus biofilms to common antifungal drugs and molecular mechanisms of antifungal drug resistance of Aspergillus biofilms. Methods The susceptibility of planktonic spores of 22 clinical isolates of Aspergillus spp. to amphotericin B, caspofungin and voriconazole was evaluated by a standard broth microdilution method. Meanwhile, in vitro model of Aspergillus spp. biofilm was established for the 22 isolates, then the susceptibility of Aspergillus spp. biofilm to amphotericin B, caspofungin and voriconazole was evaluated by a method for antifungal susceptibility testing combined with colonmetric XTT-reduction assay. In addition, real-time reverse transcription (RT)-PCR was performed to determine the relative expressions of drug efflux pump genes and azole target enzyme genes during the formation of Aspergillus spp. biofilms. Results In terms of planktonic spores of Aspergillus spp., the- minimal inhibitory concentration (MIC) range was 0.5 to 2 mg/L for amphotericin B, 0.5 to 1 mg/L for voriconazole, and the minimal effective concentration (MEC) range was 0.125 to 0.25 mg/L for caspofungin. As far as Aspergillus spp.biofilms was concerned, the sessile minimum concentration required to inhibit the growth of 50% organisms (SMIC50) and 80% organisms (SMIC80) ranged from 2 to > 32 mg/L and from 8 to > 32 mg/L, respectively, for amphotericin B, from 32 to > 256 mg/L and from 256 to >256 mg/L respectively for caspofungin, from 4 to >256 mg/L and from 32 to > 256 mg/L, respectively for voriconazole. During the formation of Aspergillus spp.biofilms, no change was observed for the expression of any of the 7 tested drug efflux pump genes or azole target enzyme genes at 4 hours, while a significant increase was noted in the expression of AfuMDRl, CYP51B and CYP51A genes at 8 hours, as well as in the expression of AfuMDRl, AfuMDR2, AfuMDR4, CYP51A,CYP51B at 12, 16 and 24 hours. Of these genes, CYP51A showed the strongest increase in expression at the above 4 time points. The expression of AfuMDR3 and atrF experienced no significant change during the formation of Aspergillus spp. biofilms. Conclusions Compared to planktonic spores, Aspergillus spp. biofilms exhibit a decreased susceptibility to amphotericin B, caspofungin and voriconazole. After the formation of biofilms, the expression of drug efflux pump genes and azole target enzyme genes is elevated in Aspergillus spp.