Poly (1,4-phenylene ether ether sulfone) (PEES) is a commonly used polymer in membrane technology for water treatment applications such as water purification and blood dialyzing in hemodialysis. In this study, PEES was chemically modified by nitration, yielding nitrated Poly (1,4-phenylene ether ether sulfone) (NPEES). Following that, NPEES nanocomposites (NCs) comprise multi-walled carbon nanotubes (MWCNTs), and the process involved the synthesis of reduced graphene oxide-oxidized single-walled carbon nanotubes, abbreviated as reduced (GO-oxSWCNTs). Various characterization techniques were used on the created membranes, such as Fourier-transform infrared spectroscopy (AT-FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) analysis. All polymer nanocomposites were found to be amorphous, according to the XRD patterns. SEM scans revealed random crater-like features on the surface of NPEES, but MWCNTs and reduced (GO-oxSWCNTs) NCs were distributed evenly on the polymer surface. The primary goal of this study was to evaluate the antimicrobial activity of modified NPEES membranes against two Gram-positive bacteria, Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis), two Gram-negative bacteria, Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli), and a fungus, Candida albicans (C. albicans). All modified membranes, including NPEES, NPEES/MWCNTs NCs, and NPEES/MWCNTs/reduced (GO-oxSWCNTs) NCs, exhibited antibacterial activity against S. aureus and B. subtilis. Notably, when compared to NPEES/MWCNTs NCs and NPEES/MWCNTs/reduced (GO-oxSWCNTs) NCs, the NPEES membrane had higher antibacterial activity, generating a 12 mm inhibitory zone. Furthermore, molecular docking studies revealed a strong fit of the tested polymer nanocomposites into the DNA gyrase B active site (PDB ID: 4uro), which was consistent with the practical results of their antibacterial activity evaluation.