The decline in the performance of spiral-wound reverse osmosis (SWRO) membranes is frequently due to biofouling. This study focus on qualitative and quantitative diagnosis of SWRO membrane biofouling. Bacterial counts on the different surfaces of the fouled membranes were carried out. Surface enhanced Raman spectroscopy (SERS) was performed to highlight clogging materials as well as their natures and identity. The topography of the fouled membranes and the structures of biofilms were visualized by fluorescence microscopy (FM) and scanning electron microscopy (SEM). The results indicated the presence of bacteria in the different SWRO membrane areas. Those strongly adhered were significantly higher than those weakly. It varied between 26 × 10 and 262 × 10 CFU m. However, SERS mapping showed different fouling levels and the thickness of the fouling layer was 5 µm. Microscopic imaging revealed biotic and abiotic deposits. These data can together allow better management of the seawater desalination process.