Effective microorganism (EM) cultures have been applied in many research fields such as agriculture, environment and bioremediation. EM is a mixed culture of microorganisms including predominant populations of lactic acid bacteria and yeasts with smaller numbers of photosynthetic bacteria, actinomycetes and other types of microorganisms. Quantitative analysis of EM is requisite for the applications of EM, as its efficiency varies depending on the composition of its main genera of EM. In this study, Rhodobacter sphaeroides, Rhodopseudomonas palustris, Lactobacillus plantarum, and Saccharomyces cerevisiae, the main genera of EM were quantified by quantitative real time polymerase chain reaction, (qRT-PCR). By using selected specific primers, photosynthetic bacteria, lactic acid bacteria and yeast were quantified with high sensitivity and specificity. The ability of viable cell count by qRT-PCR was compared with agar plate cell count, showing linear relationship. Thus, PCR based quantification system is a rapid and highly specific and sensitive tool for the quantification of EM.