The antioxidant effect of melatonin against reactive species may be modulated by its capability to alter the membrane fluidity. In this work, the antioxidant potential of the neurohormone against hydroxyl radical (^·OH) and peroxynitrite (ONOO^-) was evaluated considering its interaction with phosphatidylcholine liposomes (incorporated or not). Melatonin changes on membranes phase transitions and motional freedom degree were investigated by AFM and NMR; egg phosphatidylcholine (EPC) and dimyristoyl phosphatidylcholine (DMPC) liposomes were prepared by dialysis and extrusion methods, respectively; melatonin efflux kinetics curves were obtained through liposome destruction with Triton X-100, and monitored spectrophotometrically; ^·OH was produced from the H₂O₂ + FeCl₃ + ascorbic acid system; ONOO^- from the H₂O₂/HCl+ NaNO₂ + NaOH system; lipid peroxidation levels were detected by the TBARS method; AFM measurements were performed in liquid contact mode, in a temperature range of  16-30 °C; 81 MHz ³¹P-NMR experiments were performed at 298 K, using phosphoric acid in D₂0 as an external standard; proton spin-lattice relaxation times (T₁) were carried out at 27°C, using a t range varying from 500 a 30 e⁶ ms. The antioxidant potential of EPC liposomes-incorporated melatonin against ^·OH and ONOO^- increases about 40% and 16%, respectively. Height histograms obtained from AFM images show that the melatonin incorporated into DMPC membranes causes an inversion of lipid phase predominance from liquid crystalline to gel state, at 28°C. This suggests that the neurohormone may retard the DMPC main phase transition. ³¹P-NMR analysis suggests that melatonin induces the decrease of the motional freedom related to EPC polar groups. Despite of this result, a decrease about 18% in T₁ observed after the incorporation of melatonin into the liposomes, suggests an increase of the general membrane mobility. The melatonin fluidizing effect is probably associated with its antioxidant efficiency against ^·OH and ONOO^-.