Nutrient limitation has been proposed as a biofouling control strategy for membrane systems. However, the impact of permeation on biofilm development under phosphorus-limited and enriched conditions is poorly understood. This study analyzed biofilm development in membrane fouling simulators (MFSs) with and without permeation supplied with water varying dosed phosphorus concentrations (0 and 25 μg P·L−1). The MFSs operated under permeation conditions were run at a constant flux of 15.6 L·m2·h−1 for 4.7 days. Feed channel pressure drop, transmembrane pressure, and flux were used as performance indicators. Optical coherence tomography (OCT) images and biomass quantification were used to analyze the developed biofilms. The total phosphorus concentration that accumulated on the membrane and spacer was quantified by using microwave digestion and inductively coupled plasma atomic emission spectroscopy (ICP-OES). Results show that permeation impacts biofilm development depending on nutrient condition with a stronger impact at low P concentration (pressure drop increase: 282%; flux decline: 11%) compared to a higher P condition (pressure drop increase: 206%; flux decline: 2%). The biofilm that developed at 0 μg P·L−1 under permeation conditions resulted in a higher performance decline due to biofilm localization and spread in the MFS. A thicker biofilm developed on the membrane for biofilms grown at 0 μg P·L−1 under permeation conditions, causing a stronger effect on flux decline (11%) compared to non-permeation conditions (5%). The difference in the biofilm thickness on the membrane was attributed to a higher phosphorus concentration in the membrane biofilm under permeation conditions. Permeation has an impact on biofilm development and, therefore, should not be excluded in biofouling studies.