Highly hydrophilic and fouling-resistant polysulfone (PSF)-silica based nanocomposite ultrafiltration membranes were prepared using a one-pot method by combining phase inversion and nanoparticle formation in one step. The membrane forming solution prepared by mixing different amounts of tetraethyl orthosilicate (TEOS) with PSF solution in N-methyl-2-pyrrolidone (NMP) was cast on non-woven support and immersed into an aqueous acidic bath for phase inversion and silica nanoparticle formation. The membrane morphologies, nanoparticles distribution, hydrophilicity, and charge nature were investigated using scanning electron microscopy, atomic force microscopy, transmission electron microscopy, and contact angle as well as zeta potential measurements. The water flux, protein adsorption, and rejection were assessed, showing its applicability in filtration and separation technologies. The silica nanoparticle-containing membranes exhibited superior water permeation, rejection, and antifouling performance as compared to the neat PSF membrane. An almost 5-fold decrease in protein adsorption and a 3-fold increase in pure water flux were recorded for the nanocomposite membrane prepared with 20 wt.% TEOS. The results reveal that the membrane properties depend on the concentration of silica nanoparticles, making it possible to achieve a good trade-off between permeation and rejection.