The influence of the lateral size and the content of graphene oxide (GO) flakes in specific oxygenate functional groups on the anti-biofouling properties and performance of thin-film composite membrane (TFC) was studied. Three different multidimensional GO samples were prepared with small (500–1200 nm), medium (1200–2300 nm), and large (2300–3600 nm) size distribution, and with different degrees of oxidation (GO3 > GO2 > GO1), varying the concentration of the hydrogen peroxide amount during GO synthesis. GO1 sheets’ length have a heterogeneous size distribution containing all size groups, whilst GO2 is contained in a medium-size group, and GO3 is totally contained within a small-size group. Moreover, GO oxygenate groups were controlled. GO2 and GO3 have hydroxyl and epoxy groups at the basal plane of their sheets. Meanwhile, GO1 presented only hydroxyl groups. GO sheets were incorporated into the polyamide (PA) layer of the TFC membrane during the interfacial polymerization reaction. The incorporation of GO1 produced a modified membrane with excellent bactericidal properties and anti-adhesion capacity, as well as superior desalination performance with high water flow (133% as compared with the unmodified membrane). For GO2 and GO3, despite the significant anti-biofouling effect, a detrimental impact on desalination performance was observed. The high content of large sheets in GO2 and small sheet stacking in GO3 produced an unfavorable impact on the water flow. Therefore, the synergistic effect due to the presence of large-and small-sized GO sheets and high content of OH-functional groups (GO1) made it possible to balance the performance of the membrane.