Hygienic fecal treatment in resource-oriented sanitation (ROS) systems is an important concern. Although the addition of nitrifying microorganisms is a sustainable fecal treatment method in ROS systems, it is essential to examine the cleanliness of this method. In this study, we investigated the fate of fecal indicators in source-separated fecal samples through tracking Escherichia coli and total coliforms. The effects of adding different amounts of Nitrosomonas europaea bio-seed, along with a constant amount of Nitrobacter winogradskyi bio-seed, were studied. In intact feces samples, the pathogen population underwent an initial increase, followed by a slight decrease, and eventually became constant. Although the addition of nitrifying microorganisms initially enhanced the pathogen growth rate, it caused the reduction process to become more efficient in the long-term. In addition to a constant concentration of 10,000 cells of N. winogradskyi per 1 g feces, a minimum amount of 3000 and 7000 cells of N. europaea per 1 g feces could completely remove E. coli and total coliforms, respectively, in less than 25 days. Increasing the amount of bio-seeds added can further reduce the time required for total pathogen removal.