- Microbial load and biodegradable organic carbon were investigated in the conventional feedwater pretreatment train of the RO plant of the Alberto Pasqualini refinery, whose membranes were severely affected by biofouling. Biofilm fragments were detected in water samples from all parts of the pretreatment system. Total planktonic counts were reduced by approximately 60% in the flocculation/filtration step and remained steady at between 1.0 and 3.4 × 106 cells/mL in the remainder of the plant. Plate counts (about 104 cells/mL) were totally suppressed by free chlorine, but were restored to their original values after the activated carbon filter. DOC increased from 7 to 8 mg/L in raw water to 17 mg/L after chlorination/flocculation and remained steady at 3–4 mg/L after sand filtration. AOC of feedwater (0.9 mg/L) increased 3.5-fold after chlorination and addition of flocculants and diminished to between 1.2 and 1.6 mg/L after the sand filter. The proportion of AOC in DOC increased from 10% to 17% before sand filtration to 30–50% afterwards. BDOC assays indicated that all sections of the plant were colonized by a physiologically active microbiota, which was capable of cryptic growth. Sand, activated carbon and cartridge filters contained 5.0 × 107, 1.9 × 107 and 1.6 × 108 cells/g, respectively. A dose of the commercial biocide 30 times larger than the one used at the plant was necessary for total suppression of microbial growth in RO feedwater. The biocide did not prevent biofilm growth on RO membranes, but the fact that 30-day cleaning intervals could only be maintained by continuous biocide use indicates it affected biofilm structure. Chlorination of raw water and of pulsator sludge increased AOC by 30% and 400–600%, respectively. The organic flocculant used at the plant did not support microbial growth.