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Biofouling, as one of the membrane foulings, is the major obstacle limiting the application of polyamide (PA) thin-film composite (TFC) nanofiltration (NF) membranes in reclamation of wastewaters and treatment of industrial fluids. Herein, we introduced a facile and effective strategy for the modification of traditional PA-TFC NF membranes to improve water permeability and biofouling resistance. Specifically, three reactive guanidine molecules were successfully bonded onto the surface of PA-TFC membranes using chemical reactions between the amine groups of guanidine and the residual acyl chloride groups on the nascent PA-TFC membrane. The covalent attachment of guanidines can significantly enhance water permeability without compromising salt rejection. For instance, for 2 g L−1 Na2SO4 aqueous solution, the water flux of NFM2 increased from 25.2 L m−2 h−1 to 68.4 L m−2 h−1 at 25 °C and 0.6 MPa compared to the traditional piperazine (PIP)–trimesoyl chloride (TMC) membrane, while around 98.3%. Na2SO4 rejection was maintained. Moreover, the membranes were endowed with easy-cleaning properties, which can remarkably prevent foulant adhesion, with the water flux recovery ratio being as high as 98.9%. The developed TFC membranes also showed excellent anti-adhesive and antimicrobial activities against Gram-negative Escherichia coli K12 (E. coli), Gram-positive Bacillus pumilus LDS33 (B. pumilus) and Aspergillus parasiticus JFS (A. parasiticus). Therefore, the resultant guanidinium-functionalized NF membranes which have integrated high permselectivity and superior fouling resistance properties are promising materials for fabricating a new generation of anti-fouling/antimicrobial surfaces that are applicable to many applications such as water desalination and softening and wastewater treatment.