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A series of orthogonal array experiments were conducted using carbon source, ammonia nitrogen and total phosphorus (TP) as major influencing factors to investigate the effects of nutrients on biofouling formation and preponderant bacteria diversity in the recirculating cooling water system. Carbon source was demonstrated to be the most significant determinant affecting the biofouling formation. A minimum biofouling outcome was obtained when BOD5,NH4+-N and TP were 25, 10, and 1 mg/L, respectively. Then the preponderant bacteria strains in biofouling mass under twotypical culture conditions (negative and favorable) were identified applying both traditional biochemical methods and further molecular biology technology with phylogenetic affiliation analysis, which indicated that Enterobacteriaceae Enterobacter, Micrococcaceae Staphylococcus, Bacillaceae Bacillus, Enterobacteriaceae Proteus, Neisseriaceae Neis-seria and Pseudomonadaceae Pseudomonas were dominant under negative condition, while Enterobacteriaceae Klebsiella,Enterobacteriaceae Enteerobacter and Microbacterium-under favorable one.