- Prevention of fouling on surfaces is a major challenge that broadly impacts society. Water treatment technologies, hospital infrastructure, and seawater pipes exemplify surfaces that are susceptible to biofouling. Here we show that laser-induced graphene (LIG) printed on a polyimide film by irradiation with a CO2 infrared laser under ambient conditions is extremely biofilm resistant while as an electrode is strongly antibacterial. We investigated the antibacterial activity of the LIG surface using LIG powder in suspension or deposited on surfaces, and its activity depended on the particle size and oxygen content. Remarkably, the antimicrobial effects of the surface were greatly amplified when voltages in the range of 1.1–2.5 were applied in an electrode configuration in bacterial solutions. The bactericidal mechanism was directly observed using microscopy and fast photography, which showed a rapid bacterial movement toward the LIG surface and subsequent bacterial killing. In addition, electrochemical generation of H2O2 was observed; however, the bacterial killing mechanism depended strongly on the physical and electrical contact of the bacterial cells to the surfaces. The anti-biofilm activity of the LIG surfaces and electrodes could lead to efficient protection of surfaces that are susceptible to biofouling in environmental applications by incorporating LIG onto the surfaces.