Background: Aspergillus flavus and A. fumigatus are the leading causes of invasive and non-invasive aspergillosis. The occurrence of A. flavus is worldwide especially in tropical and subtropical regions. The vital importance of A. flavus has increased in the last years. Presently the emergence of resistance to antifungal agents among Aspergillus species is dramatically increasing. Therefore, in the present study, we evaluated the in vitro activity of five antifungal agents against A. flavus and A. fumigatus isolated from different sources.Methods: In total, 100 strains of A. flavus, which have been isolated from various specimens (nail, bronchoalveolar lavage, paranasal sinus) from suspected patients of aspergillosis patients. All strains were identified based on conventional methods and subsequently confirmed by DNA sequencing of the β-tubulin gene. The minimal inhibitory concentrations (MIC) of Amphotericin B, itraconazole, voriconazole, posaconazole and minimal effective concentrations (MEC) for caspofungin were determined using the broth micro dilution method in accordance with the guidelines of Clinical and Laboratory Standards Institute (CLSI) document M38-A2. Results: The resulting MIC90 s for A. flavus strains showed an increasing order, as follows: Caspofungin (0.031 mg/ml), posaconazole (0.25 mg/ml); voriconazole (0.5 mg/ml), itraconazole (1 mg/ml) and amphotericin B (4 mg/ml). Although MIC90 for A. fumigatus strains was different as follows: Caspofungin (0.016 mg/ml), posaconazole (0.125 mg/ml); voriconazole (0.5 mg/ml), itraconazole (0.5 mg/ml) and amphotericin B (2 mg/ml).Conclusion: The present study based on in vitro activity showed that posaconazole followed by caspofungin, voriconazole might have a potent activity, and it could be the best alternative for previous antifungal.