Amphotericin B is an antibiotic used in pharmacotherapy of life-threatening mycotic infections. Unfortunately, the applicability of this antibiotic is associated with highly toxic side effects. In order to understand molecular mechanisms underlying toxicity of amphotericin B to patients, two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of the drug and imaged with the application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. The results of the cell viability assays confirm high toxicity of amphotericin B towards human cells. The images recorded demonstrate effective binding of amphotericin B to biomembranes. Analysis of the images reveals the operation of a defence mechanism based upon the elimination of molecules of the drug from living cells via formation of small amphotericin B-containing lipid vesicles. The fact that exosomes formed are devoid of cholesterol, as concluded on the basis of the results of Raman analysis, suggests that sequestration of sterols from the lipid phase of biomembranes is not a sole mechanism responsible for the toxic side effects of amphotericin B. Alternatively, the results imply that molecules of the drug present directly within the hydrophobic membrane core disturb the lipid membrane structure and affect their biological functions.