Assessing microbiological quality assurance by monitoring bacteria in various sources of freshwater used for human consumption, recreation, and food preparation is important for a healthy life. Bacterial number and their community structure in freshwater should be determined as quickly as possible, and “real-time” and “on-site” microbiological methods are required. In this study, we examined the protocol for microchip-based terminal restriction fragment length polymorphism (T-RFLP) analysis, which uses microchip electrophoresis for rapid microbial community analysis. The availability of microchip-based T-RFLP was compared with conventional T-RFLP analysis, which uses a capillary electrophoresis system, with freshwater samples (spring water, river water, groundwater, and hydroponics solution). The detection limit of targeted bacteria by on-chip T-RFLP analysis was 1% (10³ cells/mL). The fragment sizes determined by the two analysis methods were highly correlated ( r ²=0.98). On-chip T-RFLP analysis was completed within 15 min. T-RFLP profiles of nine hydroponics solution samples were analyzed by multidimensional scaling. Considerable changes and stability in bacterial community structure during hydroponic culture were detected by both analyses. These results show that on-chip T-RFLP analysis can monitor changes in bacterial community structure, as well as conventional T-RFLP analysis. The present results indicate that on-chip T-RFLP analysis is an effective tool for rapid and “on-site” bacterial community profiling in freshwater environments, as well as freshwater used for medical and industrial purposes.