Infiuence of PTFE in the external Gas Diffusion Layer (GDL) of open-air cathodes applied tomembranelessmicrobial fuel cells (MFCs) is investigated in thiswork. Electrochemicalmeasurements on cathodeswith different PTFE contents (200%, 100%, 80% and 60%)were carried out to characterize cathodic oxygen reduction reaction, to study the reaction kinetics. It is demonstrated that ORR is not under diffusion-limiting conditions in the tested systems. Based on cyclic voltammetry, an increase of the cathodic electrochemical active area took placewith the decrease of PTFE content. This was not directly related to MFC productivity, but to the cathode wettability and the biocathode development. Low electrodic interface resistances (from 1 to 1.5 Ω at the start, to near 0.1 Ω at day 61) indicated a negligible ohmic drop. A decrease of the Tafel slopes from120 to 80mV during productive periods of MFCs followed the biological activity in the whole MFC system. A high PTFE content in the cathode showed a detrimental effect on the MFC productivity, acting as an inhibitor of ORR electrocatalysis in the triple contact zone. The lowest PTFE content (60%) manifested mechanical instability of the cathode, together with the best performance.