- Biofilm formation on surfaces in technology and the environment is a problem that can lead to high costs and can endanger human lives. Namely, infrastructure, medical implants, and food processing units, as well as oil refineries, ship hulls, and membrane technology for water treatment, are affected and underline the importance of identifying low fouling surfaces. Recently, surfaces coated with laser-induced graphene (LIG) were shown to strongly resist biofilm formation. Here we investigated the role of LIG texture and surface chemistry on biofilm formation and showed that the rough LIG surface texture correlated to enhanced biofilm inhibition. Fabrication conditions of LIG led to rough surfaces containing carbon nanofibers (250–750 nm diameter), and micropores (1–25 μm), which were shown to inhibit the attachment and proliferation of bacterial cells. In contrast, LIG surfaces with crushed nanofibers and covered micropores resisted biofilm formation less effectively. By oxidizing the LIG surface using oxygen plasma, we rendered the LIG more hydrophilic (contact angle was zero), but this chemical modification had negligible effects on the biofilm formation. After biofilm growth experiments, SEM images revealed that the morphology of Psudomonas aeruginosa cells adhered to poly(ether sulfone) substrate surfaces were rod-like in contrast to more spherical shaped cells on the LIG surfaces, which suggested distress. This study gives evidence of the importance of surface texture in antifouling applications and desirable design features such as nanofibers for effective antifouling LIG surfaces in energy, environmental, and biomedical fields.