Aims: We investigated in vitro activity of polyene drugs (amphotericin B, Amp B; and nystatin, NYS;) in combination with ethylene diamine tetra acetic acid (EDTA) against 8 pathogenic isolates of Aspergillus spp. Methods and Results: The intensity of the drug interactions was assessed using Fractional Inhibitory Concentration Index (FICI) model and Combination Index (CI) model. The interactions were further confirmed by time-kill curve study. The best combination ratio was subjected for toxicity analysis by MTT and haemolytic assay. A significant synergy was found between each of two polyenes with EDTA against Aspergillus fumigatus and Aspergillus flavus at one ratio four. The FICI ranged from 0.29 to 0.38 against A. fumigatus and A. flavus. The FICI value ranged from 0.78 to 1.26 showed no interaction against Aspergillus niger. The CI value (0.21-0.30) and time-kill curve analysis also confirmed synergistic interaction. Antagonism was not detected. There was reduction in toxicity of Amp B and NYS when used with EDTA. Conclusions: Subsequently, the chelating agent (EDTA) can be used in combination with Amp B and NYS with increased antifungal potential and reduced toxicity. Significance and Impact of the Study: To emphasize new development in antifungal drug discovery, combination therapy can be used as a tool box to expand drug development.