We investigated the bactericidal activity of bulk nanobubbles (NBs) using E. coli, a model bacterium. Bulk NBs were produced by forcing gas through a porous alumina membrane with an ordered arrangement of nanoscale straight holes in contact with water. NBs with different gas contents, including CO2, O2, and N2, were generated and evaluated for their bactericidal effects. The survival rate of E. coli was significantly reduced in a suspension of CO2-containing NB (CO2-NB water). The N2-NB water demonstrated a small amount of bactericidal behavior, but its impact was not as significant as that of CO2-NB water. When E. coli was retained in O2-NB water, the survival rate was even higher than that in pure water (PW). We investigated the generation of reactive oxygen species (ROS) in NB suspensions by electron spin resonance spectroscopy. The main ROS generated in the NB water were hydroxyl radicals and OH·, and the production of ROS was the strongest in CO2-NB water, which was consistent with the results of the bactericidal effect measurements. We assumed that NB mediated by ROS would exhibit bactericidal behavior and proposed a kinetic model to explain the retention time variation of the survival rate. The results calculated based on the proposed model matched closely with the experimental results.