Biofouling development is affected by a variety of factors that change over the length of reverse osmosis (RO) membrane modules in pressure vessels. Spatially resolved biofouling formation was studied under conditions representative to practice using four one-meter Long Channel Membrane Test Cells (LCMTCs) in series, simulating an industrial pressure vessel. Biofouling was induced by dosing an easily assimilable substrate to the feed water. The impact of biofouling on the sequential decline of RO membrane performance indicators (feed channel pressure drop, permeability and salt rejection) was investigated. Also, the temporal organic carbon (DOC) consumption was assessed spatially over the four test cells. Results showed that all membrane performance indicators were impacted by biofouling formation. The feed channel pressure (FCP) drop increase was impacted earliest and strongest followed by permeability and salt rejection decline, underlining that FCP drop is a sensitive and early biofouling monitoring indicator. Spatially resolved biofouling investigations revealed that most biofouling was formed in the lead sections of membrane installation with a decreasing gradient over length, linked to DOC availability in the system. In this study, FCP drop played a crucial role: the FCP drop increase at the lead test cell of the membrane installation caused performance losses for the downstream test cells. Minimizing the effect of biofouling on membrane performance should be pursued by a combination of strategies involving (i) early detection and preventive cleaning, (ii) substrate limitation for delaying biofouling built-up and (iii) optimized (early) cleaning procedures for more effective biofilm removal.