Water transport through the cathode and oxygen presence/absence in the anodic solution, are important for operation and performance of single chamber microbial fuel cells (SCMFCs). This study focused on water transport and biofilm formation on carbon papers with different characteristics: hydrophobicity, thickness, and presence of a micro porous layer (MPL). The results showed that higher hydrophobicity, thicker structures and the presence of the MPL decreased the water transport over time. The carbon papers with low hydrophobicity had complete penetration of biofilms and higher water transport, while those with high hydrophobicity and MPL had no biofilm penetration and lower water transport, indicating a clear correlation of biofilm penetration and water transport across carbon papers. Salt precipitation on carbon papers clogged the pores and resulted in lower water transport. Cyclic voltammograms using Au/Hg microelectrode inserted near the cathode indicated the absence of oxygen in the anodic solution due to the oxygen consumption by aerobic/anaerobic biofilms on cathodes. The results of water transport and oxygen presence illustrated that the biofilms can be used as a cost-effective membrane/separator for SCMFCs.