A method of esterifying polyene microlide antibiotics having a carboxyl group and selected from the group consisting of nystatin, aureofacin, candicidin, pimaricin, amphotericin B and polyfungin and the N-substituted acetyl and N-penten-2-on-3-yl-2 derivatives of these antibiotics, which comprises the steps of reacting said antibiotic compound with an alcohol selected from the group consisting of one to five carbon aliphatic alcohols and monocyclic aryl alcohols;
- in at least one neutral organic solvent therefor;
- in the presence of a reaction promotor selected from the group consisting of dicyclohexyl carbodiimide and mixtures of dicyclohexyl carbodiimide with hydroxybenzatriazole;
- at temperatures up to 40.degree. C. until completion, isolating said ester and then purifying same.