Fouling caused by organic matter and bacteria remains a significant challenge for the membrane-based desalination industry. Fouling decreases the permeate quality and membrane performance and also increases energy demands. Here, we quantified the amount of organic matter and bacteria at several stages along the water-treatment train of an integrated ultrafiltration–nanofiltration seawater treatment pilot plant. We quantified the organic matter, in terms of Total Organic Carbon (TOC) and Assimilable Organic Carbon (AOC), and evaluated its composition using Liquid Chromatography for Organic Carbon Detection (LC-OCD). The bacterial cells were counted using Bactiquant. We found that ultrafiltration (UF) was effective at removing bacterial cells (99.7%) but not TOC. By contrast, nanofiltration (NF) successfully removed both TOC (95%) and bacterial cells. However, the NF permeate showed higher amounts of AOC than seawater. LC-OCD analysis suggested that the AOC was mostly composed of low molecular weight neutral substances. Furthermore, we found that the cleaning of the UF membrane using chemically enhanced backwash reduced the amount of AOC released into the UF permeate. By implementing the cleaning-in-place of the NF membrane, the pressure drop was restored to the normal level. Our results show that the UF and NF membrane cleaning regimes investigated in this study improved membrane performance. However, AOC remained the hardest-to-treat fraction of organic carbon. AOC should, therefore, be monitored closely and regularly to mitigate biofouling in downstream processes.