Conducting Nafion/SiO2 composite membranes were successfully prepared using a simple electrostatic self-assembly method, followed by annealing at elevated temperatures of 240, 270, and 300 °C. Membrane performance was then investigated in vanadium redox flow batteries (VRB). These annealed composite membranes demonstrated lower vanadium permeability and a better selectivity coefficient than pure Nafion membranes. The annealing temperature of 270 °C created the highest proton conductivity in the Nafion/SiO2 composite membranes. The microstructures of these membranes were analyzed using transmission electron microscopy, small-angle X-ray scattering, and positron annihilation lifetime spectroscopy. This study revealed that exposure to high temperatures resulted in an increase in the free volumes of the composite membranes, resulting in improved mechanical and chemical behavior, with the single cell system containing composite membranes performing better than systems containing pure Nafion membranes.