In this study, a simple method based on non-ionic surfactant polysorbates-80 was used to create mesoporous γ-Al2O3NPs. The properties of the prepared mesoporous alumina nanoparticles (Al2O3NPs) were verified using ATR-FTIR, XRD, SEM, TEM, DLS, and BET surface area analysis. Then, thin-film nanocomposite (TFN) nanofiltration membranes were fabricated by interfacial polymerization of embedded polyamide layers with varied contents (0.01 to 0.15 wt.%) of mesoporous γ-Al2O3NPs. The surface roughness, porosity, pore size, and contact angle parameters of all the prepared membranes were also determined. The performance of the fabricated membranes was investigated under various mesoporous γ-Al2O3NPs loads, time, and pressure conditions. Mesoporous γ-Al2O3NPs revealed an important role in raising both the membrane hydrophilicity and the surface negativity. The addition of 0.03 wt.% mesoporous γ-Al2O3NPs to the TFN membrane increased water flux threefold compared to the TF control (TFC) membrane, with maximum water flux reaching 96.5, 98, 60, and 52 L/(m2.h) for MgSO4, MgCl2, Na2SO4, and NaCl influent solutions, respectively, with the highest salt rejection of 96.5%, 92.2%, 98.4%. The TFN-Al2O3 membrane was also able to soften water and remove polyvalent cations such as Mg2+ with a highly permeable flux. The TFN-Al2O3 membrane successfully removed the hardness of the applied water samples below the WHO limit compared to using merely the TFC membrane. Furthermore, the TFN-Al2O3 nanofiltration membrane unit proved to be a promising candidate for the desalination of real brine like that collected from the Safaga area, Egypt.