To curtail the potential of donor corneal tissue disseminating fungi to the recipient's eye, we evaluated the addition of amphotericin B to McCarey-Kaufman (M-K)-corneal storage medium supplemented with colistin. Amphotericin B was examined for its ability to inhibit the growth of Candida albicans and Aspergillus flavus using a microbroth dilution test and checkerboard assay in combination with only gentamicin and a combination of colistin, gentamicin, and amphotericin B. The safety on epithelium and endothelium was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The minimal inhibitory concentration of gentamicin was found to be >256 μg/ml against both C. albicans and A. flavus, whereas that of amphotericin B was found to be in a range of 0.25-0.5 and 1-2 μg/ml for C. albicans and A. flavus, respectively. According to the checkerboard assay, 80% (4/5) of C. albicans isolates and 100% (5/5) of A. flavus isolates responded synergistically to the combination of amphotericin B and gentamicin, but only 20% (1/5) of C. albicans isolates showed an additive effect. None of the tested isolates displayed antagonism. The combined effect of the three drugs also did not display any antagonistic effect. Additionally, the MTT assay reveals no toxic effect of the antimicrobials used on corneal epithelial and endothelial cells. In vitro experiments demonstrate that amphotericin B is not toxic to either epithelium or endothelium and is a promising additive to the M-K medium supplemented with colistin.