A study of the pro‐oxidant capacity of amphotericin B (AMP) was carried out by analyzing the photosensitizing ability of an AMP model. Although AMP is a well‐known broad‐spectrum antibiotic, little is known about the mechanism of its pro‐oxidant capability. This study employed density functional theory (DFT) to assess the photosensitizing capacity of AMP. Furthermore, electron transfer reactions were modeled, considering that lipid media can mimic the environment of the cell membrane. The results show that AMP is a pro‐oxidant molecule mainly through Type II mechanism, with the corresponding production of 1O2. In this way, AMP could induce oxidative stress because it is capable of generating reactive oxygen species. In this case, AMP showed a reaction rate constants of 8.94 × 109 and 1.89 × 1010 M−1 s−1 for Type I and II mechanisms, respectively, whereas the overall reaction rate constant was 2.79 × 1010 M−1 s−1. Therefore, the results of this study support the pro‐oxidant capacity of AMP in lipid media, and this mechanism could contribute to the antifungal activity of AMP.