In this study, multiparametric flow cytometry (FCM) was installed to enumerate the diagnosis of Pseudomonas aeruginosa ATCC 10145 and Escherichia coli K12 (IFO 3301). The nucleic acids (DNA/RNA) were double stained by a LIVE/DEAD bacLight viability kit, involving green SYTO 9 and red propidium iodide (PI), based on the permeability of two chemicals according to the integrity of plasma membrane. As the results showed, the gate for dead bacteria was defined as the range of $0.2{ imes}10^0$ to $6.0{ imes}10^1$ photo multiplier tube (PMT) 2 fluorescence (X-axis) and $2.0{ imes}10^0$ to $2.0{ imes}10^2$ PMT 4 fluorescence (Y-axis), and the gate for live bacteria was defined as the range of $6.0{ imes}10^0$ to $6.0{ imes}10^2$ PMT 2 fluorescence (X-axis) and $2.0{ imes}10^0$ to $4.0{ imes}10^2$ PMT 4 fluorescence (Y-axis). In the comparison of the number of the tested bacteria detected by FCM (viability assessment) and plate culture (cultivability assessment), the number of bacteria detected by FCM well represented the number of bacteria that was detected by the colony forming unit (CFU) counting method when bacteria were exposed to isopropyl alcohol and silver/copper cations. Consequently, it is concluded that the application of FCM to monitor the functional effect of disinfectants on the physiological status of target bacteria can offer more rapid and reliable data than the plate culture colony counting method.