A simple and rapid method was developed for monitoring of Gram-positive bacteria in the wastewater treatment system. Culture suspensions of 4 Gram-positive and 4 Gram-negative strains were filtrated and stained with a polyethersulfone membrane filter and Toluidine Blue-O. To establish quantitative color image analysis, the intensity value of RGB (red-green-blue) color of a scanned filter image was analyzed with a photographic program. Red and green color values of Gram-positive bacteria were higher than those of Gram-negative bacteria. This method was applied to the activated sludge mixed with the Gram-positive bacteria. Although evaluation was difficult due to the irregular size and shape of flocs, the population of Gram-positive bacteria in the activated sludge could be monitored with floc dispersion technique. The more amounts of Gram-positive bacteria in the activated sludge led to the increase of red and green color values. This method provides a rapid and quantitative measurement of Gram-positive bacteria within the wastewater treatment systems.