The separation of biomacromolecules, mainly proteins, plays a significant role in the pharmaceutical and food industries. Among the membranes' techniques, thin-film nanocomposite nanofiltration membranes are the best choice due to their high energy efficiency, excellent productivity, cost-effective and tuneable properties that have captured the attention of the efficient separation of biomacromolecules, especially from the industrial perspective. The present work directs the efficient separation study of proteins, namely, lysozyme, trypsin, pepsin, bovine serum albumin (BSA), and cephalexin, using a thin-film nanocomposite membrane integrated with Arg-MMT (argininemontmorillonite) clay nanoparticles. The surface morphology and cross-section images of the TFN membranes were studied using a field emission scanning electron microscope (FE-SEM) and a high-resolution transmission electron microscope (HR-TEM). The thermal stability and hydrophilicity of the membranes were examined using thermogravimetric analysis (TGA) and contact angle, respectively. The surface chemistry of the selective layer has different functional groups that were analyzed using FTIR spectroscopy. The performance of the membranes was studied at different trans-membrane pressures and permeation times. The effect of monomer concentration on the separation performance of the membranes was also studied at different permeation times. The membranes' antibacterial activity was evaluated using the Muller− Hinton disk diffusion method using gram-negative Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus) bacteria. The highest rejection was achieved for BSA up to 98.92 ± 1%, and the highest permeation was obtained against lysozyme feed solution up to 26 L m −2 h −1 at 5 bar pressure. The membrane also illustrated excellent rejection of cephalexin antibiotics with a rejection of 98.17 ± 1.75% and a permeation flux of 26.14 L m −2 h −1 . The antifouling study performed for the membranes exhibited a flux recovery ratio of 86.48%. The fabricated thin-film nanocomposite membrane demonstrated a good alternative for the separation of biomacromolecules and has the potential to be used in different sectors of industry, especially the pharmaceutical and food industry.