Activated Sludge Membrane Bioreactors (AS-MBR) are recognized as a commercially competitive alternative to conventional wastewater treatments. However, membrane fouling remains one of the main challenges and disadvantages of the process. This study evaluates the suitability of Optical Coherence Tomography (OCT) in monitoring the cake layer development in-situ in AS-MBR under continuous operation. Real-time direct imaging of the cake layer was feasible when limiting the continuous movement of the AS flocs in the reactor by turning aeration off for few minutes prior to scanning a given membrane area. The cake layer morphology was evaluated using both 2D and 3D image analysis. The 3D analysis respect to 2D analysis provided a more representative characterization of the fouling formed in the system. The non-invasive nature of OCT imaging enabled monitoring fouling development over time, where an increase in thickness and a decrease in roughness was observed in the first 200 h of operation. The 3D OCT image analyses were also compared with the 3D confocal laser scanning microscopy (CLSM) image analyses performed at the end of the study. Results demonstrate that OCT imaging can be applied for online, real-time monitoring and analysis of fouling behavior in AS-MBR systems.