The motivation in this research is that Berberine’s antifungal mode of action is not known yet. The focus of this study is to identify the biochemical basis of yeast sensitivity to berberine (i.e. "mode of action"). The MIC of berberine with yeast was determined to be in the range of 0.156 to 0.078 mg/ml. To infer berberine mode of antifungal activity, GDA analysis was performed using a library of 4700 yeast strains, each with a different gene deletion, using a berberine concentration of 0.13 mg/ml. Out of the library, the 71 most sensitive yeast deletion mutant strains and 22 resistant strains were identified and analyzed with GO terms. Finally, using berberine’s natural florescence characteristic together with other histochemical stains, confocal fluorescence microscopy revealed that berberine was dynamically localized first to mitochondria and then to oil droplets within the yeast cell.