The aim of this study was the fabrication of polysulfone membranes with antimicrobial properties. The strategy employed was based on the use of carbon nanofibers (CNF) presenting carboxyl functional groups (CNF-COOH) and silver nanoparticles (AgNPs). The materials were investigated by XPS, SEM, TEM, EDX, and ICP-MS. The antibacterial activity was ascertained towards Escherichia coli and Bacillus subtilis both in solid and liquid media employing different synthesis routes for the AgNPs. In solid phase the Gram-positive bacteria showed higher sensitivity for PSF-CNF-Ag membranes, while in liquid phase the antimicrobial activity of the hybrid membrane is more pronounced towards Gram-negative species. Furthermore, in the case of E. coli, the growth inhibition in liquid medium is probably due to the synergetic action of the modified CNF and AgNPs.