Microbial fouling is a serious problem in open recirculating cooling water systems. The bacterial communities that cause it have not been fully understood. In this study, we analyzed the community structure of free-living bacteria and particle-attached bacteria in cooling water, and bacteria in biofilm collected from the wall of the water reservoir in an industrial recirculating cooling water system by construction of a 16S rRNA gene clone library. Based on amplified ribosomal DNA restriction analysis, clones of all three libraries were clustered into 45 operational taxonomic units (OTUs). Thirteen OTUs displaying 91–96% sequence similarity to a type strain might be novel bacterial species. Noted differences in community structure were observed among the three libraries. The relative species richness of the free-living bacteria in cooling water was much lower than that of particle-attached bacteria and bacteria in biofilm. The majority of the free-living bacterial community (99.0%) was Betaproteobacteria. The predominant bacteria in the particle-attached bacterial community were Alphaproteobacteria (20.5%), Betaproteobacteria (27.8%) and Planctomycetes (42.0%), while those in the biofilm bacterial community were Alphaproteobacteria (47.9%), Betaproteobacteria (11.7%), Acidobacteria (13.1%) and Gemmatimonadetes (11.3%). To control microbial fouling in industrial recirculating cooling water systems, additional physiological and ecological studies of these species will be essential.