Biofouling in membrane filtration systems poses significant operational challenges, leading to decreased permeate flux. The aim of this work was to study the anti-biofilm properties of new nanofiltration membranes produced via layer-by-layer, LBL, assembly by coating a polyvinylidene fluoride (PVDF) support with a polyethylenimine (PEI) and poly(acrylic acid)/graphene oxide (PAA-GO) mixture. The membranes were characterized according to contact angle, scanning electron microscopy (SEM), atomic force microscopy and their Z-potential. Biofilm quantification and characterization were carried out using crystal violet staining and SEM, while bacterial viability was assessed by using colony-forming units. The membrane with three bilayers ((PAA-PEI)3/PVDF) showed a roughness of 77.78 nm. The incorporation of GO ((GO/PAA-PEI)3/PVDF) produced a membrane with a smoother surface (roughness of 26.92 nm) and showed salt rejections of 16% and 68% for NaCl and Na2SO4, respectively. A significant reduction, ranging from 82.37 to 77.30%, in biofilm formation produced by S. aureus and E. coli were observed on modified membranes. Additionally, the bacterial viability on the modified membranes was markedly reduced (67.42–99.98%). Our results show that the modified membranes exhibited both antibiofilm and antimicrobial capacities, suggesting that these properties mainly depend on the properties of the modifying agents, as the initial adherence on the membrane surface was not totally suppressed, but the proliferation and formation of EPSs were prevented.