Laser-induced graphene (LIG) is a graphitic material that can be easily formed by CO2-laser irradiation on a variety of carbon-based substrates, including porous polymeric membranes. LIG-membrane formation involves lasing the top surface of a polymeric membrane, and converts essential polymer separation structures to porous LIG foam. However, in previous studies, separation properties could be partly recovered by forming LIG polymer composite layers or by coating the polymeric membrane substrate with trimethylaluminum (TMA) or graphene oxide (GO) to avoid melting or damaging the membrane pore structure and the subsurface polymeric structures during the lasing process. Herein, by optimizing the laser settings and fabrication conditions, we made LIG directly on uncoated porous PES membranes, while preserving the subsurface polymer. The effects of lasing on membrane properties were studied by comparing porous polymeric PES membranes fabricated using the non-solvent induced phase separation (NIPS) or the vapor-induced phase separation (VIPS) method. Membrane fabrication conditions such as the polymer concentration of the casting solution and the exposure time to the non-solvent were varied, and the NIPS method resulted in membranes with a finger-like polymer substructure morphology while the VIPS method resulted in membranes with an asymmetric cellular morphology. LIG-membranes prepared on NIPS membranes resulted in large permeability changes, while LIG on VIPS membranes gave only very minor changes. The antimicrobial activity of these LIG-membranes as porous electrodes was dependent on applied voltage and solution contact time and 4–6 log removal of bacteria at 10 V was achieved. Understanding LIG formation on porous polymeric membranes will minimize processing steps and might lead to electrically conductive membranes with controlled separation properties.