The study investigated the environmental and pretreatment impact on biofouling in a seawater reverse osmosis (SWRO) plant. The effect of a pretreatment chemical (chlorine) and certain alterations of chemical dosing on membrane biofouling was also investigated, as well as the biofouling potential of the source water and the effect of chlorination on this biofouling potential. Experiments were carried out on biofouling in a SWRO plant on the Red Sea coast with a set of four pretreatment modes: (1) normal operation mode, where coagulant is dosed immediately before the media filter and where sodium metabisulfite (SBS) is dosed after the media filter; (2) operation with the coagulant dosing point shifted back to the pressure side of the seawater intake pump; (3) with the SBS dosing point shifted to after the micron cartridge filter; (4) while the plant was operating without chlorination/dechlorination. Bacterial generation time and biofilm attachment slides were used to evaluate biofouling. Generation (doubling) times were lowest (higher multiplication capacity) nearest the intake, and they increased gradually along the pretreatment line, becoming the highest closest to the membranes and in the brine reject. When the SBS was shifted, chlorine removal became closer to the membranes. Following this, generation time in the water samples taken after the dual media filter (ADMF), after the micron cartridge filter (AMCF) and immediately before the membranes, decreased significantly, reflecting more biofouling potential in the membranes. This correlates well with operational data where the SBS shift resulted in doubling the frequency of membrane cleaning. Generation times were higher when no chlorine was used, indicating less membrane biofouling potential. Water samples from the plant's intake in the sea had 24-h generation time values less, but close to those of chlorinated seawater. This indicated high nutrient load and questionable water quality of the intake. The bacterial attachment to the biofilm slides showed the general trend exhibited by the generation time of planktonic bacteria. The biofilm formation in the brine was generally less than the preceding sampling stations ADMF and AMCF, indicating removal of nutrients along the pretreatment line, However, when the SBS dosing point was shifted closer to the membranes, the number of attached bacteria in the brine increased significantly. Bacteria attached to the biofilm slides randomly and in microcolonies with vesicles in between. Although chlorination enhances biofouling, the current experiments indicate that the questionable quality of source water is one cause of the operational problems in this plant.