A new crystallization method of N-iodoacetylamphotericin B derivative is reported. The crystallization process in the presence of different quantities of amphotericin B additives was extensively studied and its mechanism was proposed. It also resulted in good quality single crystals suitable for X-ray structure determination (100 K). The structural information obtained allowed for periodic and dimer single point computational studies at the B3LYP/6-31G* level of theory. These confirmed the proposed controlled crystallization mechanism and the stabilization of crystals formed by the iodoacetyl derivative and parent amphotericin B. The calculation results indicate the strength of different intermolecular interactions and reveal the great contribution of the solvent molecules to the crystal lattice formation, with the total energy gain of about 335 kJ•mol -1, which almost doubles the cohesive energy value. Hirshfeld surface analysis shows the more efficient crystal packing of N-iodoacetylamphotericin versus amphotericin B and the effect of the iodoacetyl group on the intermolecular contacts. The generated electrostatic potential maps reveal the impact of the iodoacetyl substituent on the nitrogen atom basicity and thus confirm the stronger hydrogen bonding created via nitrogen atom in the case of N-iodoacetyl amphotericin B, and higher drug activity of amphotericin B related to the ability of the zwitterion formation.