This paper presents an experimental study of the inactivation in water of two representative classes of bacteria, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, using pulsed dielectric barrier discharges (PDBDs) in a coaxial arrangement. To this purpose, an adjustable plasma source supplies 25-kV/500-Hz pulses, 30 μs long, at atmospheric pressure, with total energy consumption estimated at about 100 mJ/pulse. The inactivation effect of a PDBD on these types of microorganisms has been previously studied in dependence on an oxygen gas flow mode (null, continuous, and modulate). The results have shown a significant bacterial reduction rate from 108 to 103 cells/mL with E. coli and from 107 to 103 cells/mL with B. subtilis. The inactivation effectiveness is substantially similar in both kinds of bacteria, although some data suggest a greater susceptibility of the Gram-negative E. coli to plasma exposure. Plasma diagnostics was carried out using optical emission spectroscopy whereby the OH radical and reactive oxygen species formation rates in solution were found and the level of ozone produced by the discharge was monitored. Finally, a kinetics model was developed to characterize the chemical species taking place in nonthermal plasma inactivation processes in water.