Abstract: The effect of melatonin was evaluated on three phosphatidylcholine‐based membrane models. Changes in liposome dynamics were monitored by fluorescence, following the response of the probe merocyanine‐540, as well as by differential scanning calorimetry (DSC). Langmuir monolayers were investigated using molecular area measurements, as well as by Brewster angle microscopy (BAM). Mica‐supported bilayers were observed via atomic force microscopy (AFM). Fluorescence results demonstrating that melatonin increases the affinity between MC‐540 and lipid molecules possibly because of an increase in the membrane fluidity in liposomes. DSC analyses showed that melatonin promoted a reduction in enthalpy in the lipid nonpolar chains. Melatonin also promoted an increase in the molecular area of Langmuir monolayers, as well as a decrease in membrane thickness. Consequently, melatonin appeared to induce re‐ordering effects in liposome and Langmuir monolayers. AFM images of bilayers immobilized on mica suggested that melatonin induced a gel state predominance or a delay in the main phase transition. At experimental conditions, melatonin interacted actively with all membranes models tested and induced changes in their physico‐chemical properties. The data presented here may contribute to the understanding of melatonin physiologic properties, as well as the development of therapeutic advanced systems, such as drug delivery systems and biosensors.