Amphotericin B (AmB) is an antifungal polyene for which the most accepted mode of action is formation of protein-like ion channels in the cell membrane. Patch-clamp research on Candida albicans protoplasts carried out in the outside-out configuration showed that application of 0.05 and 0.1 μM AmB caused a decrease in seal resistance. Such a phenomenon can be correlated with a decrease in membrane tightness. AmB applied at a 0.05 μM concentration also caused a decrease in the number of active TOK1 (two-pore outward rectifiers) potassium channels, but did not significantly change their open probability. The results indicate that in C. albicans protoplast AmB causes a decrease in cell membrane integrity by interaction with its lipid phase but not with ion channels. Fluorescence microscopy techniques showed that AmB treatment, in clinical concentrations, had no effect on the percentage of PI-positive protoplasts. AmB treatment in the concentrations tested did not cause a rapid reduction of the number of C. albicans protoplasts. However, there was a significant loss of replication competency and numerous morphological and physiological disorders, including cytoplasm shrinking, abnormal morphology of the nucleus and mitochondria, a sudden decrease in the MTT reduction level and oxidative stress. Our results show that the induction of yeast cell death by AmB, at therapeutic doses, is a multistage and long-term process involving multiple intracellular pathways.