The paper presents the results of the first spectroscopic, microcalorimetric, and crystallographic study analyzing in great detail the strongly synergistic composition containing a selected 1,3,4-thiadiazole derivative: 4-(5-methyl-1,3,4-thiadiazol-2ylo)benzene-1,3-diol (C1) and the antibiotic amphotericin B (AmB) in model biological DPPC films additionally modified with sterols: cholesterol (Chol) and ergosterol (Erg). The spectroscopic properties of the analyzed composition were studied with the use of spectroscopic methods, including: measurements of electronic fluorescence and absorption spectra with the technique of resonance light scattering (RLS), measurements of stationary fluorescence anisotropy and time-resolved fluorescence lifetimes with the method of single photon counting (TCSPC), circular dichroism spectra (CD), and infrared FTIR spectra. The mentioned methods were further complemented by including microcalorimetric DSC and crystallographic XRD analyses. All themeasurements were taken in model biological systems formed as liposomal films and liposomal multilayers composed of DPPC and/or modified with sterols: cholesterol and ergosterol. The absorption spectra measured for C1/AmB and the synergistic composition thereof revealed differences indicating that while the antibiotic forms retained its known tendency to form aggregate in film systems, the synergistic composition had certain effects on the aggregational equilibrium. This was also reflected in the fluorescence emission measured for said systems, in particular the RLS and CD spectra, as well as, to an extent, the results of fluorescence anisotropy measurements. As we proceeded to the analysis of FTIR spectra, it was observed that the synergistic composition of C1 + AmB showed stronger interaction with the hydrophobic layer of the film. In turn, the crystallographic measurements performed for the synergistic composition revealed its impact on the multilayer's thickness. Finally, microcalorimetric measurements indicated that the synergistic composition had a lesser impact on the main phase transition temperature of the lipid, as compared to the respective compounds tested separately. The presented paper is the most detailed report to date pertaining to the synergism observed for the relevant systems analyzed in model biological films modified with sterols. The study provides an in-depth spectroscopic, microcalorimetric, and crystallographic description of the molecular interactions analyzed for said molecular systems in model films formed of DPPC with the addition of sterols. Its results are particularly significant given the fact that AmB continues to be a clinically relevant antibiotic employed in the treatment of particularly severe internal mycoses and against otherwise resistant fungal strains.