Membrane-aerated biofilm reactors (MABRs) are a novel technology based on the growth of biofilms on oxygen-permeable membranes. Hereby, MABRs combine all the advantages of biofilm growth with a more flexible and efficient control of the oxygen load. In the present work, flow cell operation to achieve full nitrification revealed a significantly different structure of nitrifying MABR biofilms with respect to its co-diffusion counterparts reported in the literature (up to now assumed to have similar properties). Different levels of shear stress and oxygen loadings during MABR operation also affected these biofilm parameters. Furthermore, reactor operation at higher oxygen loads resulted in an increase of the biofilm cohesiveness, which depended on the EPS mass in the biofilms and the type of stress applied (more cohesive against normal than shear stresses). The EPS in the strongest biofilms had a higher content of proteins and a lower level of carbohydrates. Staining analyses revealed that the EPS in the stronger biofilm regions had hydrophilic nature and distributed around dense microbial aggregates, whereas it was homogeneously distributed in the weaker strata. Overall, the obtained results provide input parameters to future modelling efforts and operating conditions to support more robust autotrophic N conversions in MABRs.