Membrane fusion is one of the most important processes for the survival of eukaryotic cells and entry of enveloped viruses to the host cells. Lipid composition plays a crucial role in the process by modulating the organization and dynamics of the membrane, as well as the structure and conformation of membrane proteins. Phosphatidylethanolamine (PE), a lipid molecule with intrinsic negative curvature, promotes membrane fusion by stabilizing the non-lamellar intermediate structures in the fusion process. Conversely, oleic acid (OA), with intrinsic positive curvature, inhibits membrane fusion. The current study aimed to investigate polyethylene glycol-mediated lipid mixing, content mixing, content leakage, and depth-dependent membrane organization and dynamics, using arrays of steady-state and time-resolved fluorescence techniques, to determine the causative role of PE and OA in membrane fusion. The results demonstrated that the presence of 30 mol % PE in the membrane promotes membrane fusion through a mechanism that circumvents the classical stalk model. On the contrary, membranes containing OA showed reduced rate and extent of fusion, despite following the same mechanism. Collectively, our findings in terms of membrane organization and dynamics indicated a plausible role of PE and OA in membrane fusion.