The type and loading level of oxygen-containing functional groups on graphene oxide (GO) nanosheets significantly affect the size and alignment of nanochannels formed between the GO nanosheets and the separation performance of laminar GO membranes. Here, we demonstrate how double-oxidation of GO leads to the higher surface charge of GO nanosheets, the formation of highly stable water-based GO solution, more-ordered deposition of GOs on the polyethersulfone membrane through the pressure-assisted self-assembly method, and the formation of highly durable GO membranes possessing smoother surface morphology and higher antifouling properties. A multi-technique investigation was applied to follow the physicochemical difference between GO and double-oxidized GO, and the physical stability and separation performance of the corresponding membranes using experimental and computational studies. The double-oxidized GO-based membranes provided a significantly high water flux of 230 L/(m².h) in 2.5 bar transmembrane pressure, excellent rejection of 99.9% for methylene blue (MB) dye, and outstanding separation performance stability over time. In contrast, GO membranes showed rejection of 81.5% for MB, and their separation performance diminished significantly over time. The antifouling properties of double-oxidized GO membranes were substantially higher (∼ four times) due to their higher negative surface charge and smoother surface morphology. The density functional theory (DFT) was used to gain insight into the interactions between the functional groups and the reasoning for the higher mechanical stability of double-oxidized GO membranes. Results revealed that the formation energy of GO decreases by increasing the number density of functional groups. It was also found that a higher number of carboxyl groups at the edges of the double-oxidized GO leads to higher hydrogen bonding, higher binding energy, and a more stable GO-membrane structure.