This study was focused on choosing a nucleic acid fluorescent dye for use in enumerating dead and living bacteria in cultured milk. Ultra-high temperature sterile milk was inoculated with a mixed strain lactococcal starter culture and incubated overnight at 37°C. A negative control containing only dead cells was obtained by heating the cultured milk at 85°C for 12 min. An aliquot of milk was then diluted 500-fold in PBS buffer, and then one of 5 nucleic acid dyes was added (Sybr Green, Syto 9, Syto 24, carboxy fluorescein diacetate (cFDA) or Thiazole Orange) in combination with propidium iodide. Fluorescence from binding of the dyes in living bacterial cells was detected at emission wavelength of 488 nm. Fluorescence from PI that had permeated into dead bacterial cells and quenched fluorescence from the other dyes was detected at 640 nm. Fluorescence from cFDA only occurs upon intracellular enzymatic metabolism after its permeation into living cells. The nonheated cultured milk contained both living and dead cells as shown by their position on the cytograph. After heating the cultured milk, only dead cells were detected. Sybr green performed better in distinguising between live and dead populations for assessment of viability of bacterial cells in cultured milk. Flow cytometery measurements using Sybr green were more reproducible than when using the other dye stained samples. Between the Syto dyes, Syto 24 exhibited more consistent results in terms of bacterial enumeration. A problem with the method was that diluting the milk so as not to clog the flow cytometer makes the background noise more prominent compared with the fluorescent from the bacterial cells. Flow cytometric based enumeration is a promising tool yet requires additional studies.