Rapid and continuous pressure treatment was realized using a hydraulic pump and the momentary decompression following high pressurization was used to inactivate bacteria. The number of colony-forming E. coli decreased to 1/1000 in response to 10 cycles of pressure treatment. In groundwater samples, repeated pressure treatment led to a two-log decrease in the number of colony-forming bacteria. These findings suggest that repeated cycles of momentary decompression following high pressurization enabled a marked decrease in bacterial growth activity. The results presented herein may contribute to microbiological quality control and the safety of freshwater.