Interaction of primycin antibiotic with oleic acid was investigated to understand the effect of primycin on lipid membranes at a molecular level. The thermodynamic parameters of the complex formation were determined by photoluminescence studies. Results highlight the presence of two interactions between the interacted species according to the cases in which one or two hydrogen bonds stabilize the molecular complexes. Although both interactions result in similar Gibbs-free enthalpy change at room temperature, the enthalpy and entropy changes associated with the formation of single or double hydrogen bonds are quite different. Quantum chemical and anisotropy decay studies validated that the formation of a single or double hydrogen bond between these species is driven by entropy in the former or enthalpy in the latter case. Owing to the resultant quite different temperature dependence of these two interactions, after detailed examinations in a cellular environment, this property could have importance in application of primycin on differently fevered bodies.