Ciliated protozoa play many fundamental roles in the aquatic environments where they thrive. They mediate the microbial loop of the universal food web, control the surrounding organisms, and act as biomarkers for water quality, beside some capability for the production of bioactive compounds. Identification of different ciliate species is chiefly based on morphological taxonomy. In order to develop molecular tools that facilitate the exploration of freshwater Nile ciliates, a direct, 18srDNA-based PCR assay was tuned up. This assay was tested for DNA-barcoding of some species that prevail the Nile in Mansoura City in the North of Egypt. The resulting sequences were applied to design new species-specific PCR assays for detection of density of these ciliates in River Nile. Using both newly-designed primers and assays, the top identified species were Euplotes woodurffi, Halteria grandinella, and Coleps hirtus. The species-specific primers produced variable-sized PCR amplicons, with densities that were at least correlated by 95 % with the real species count in the riverine water. The newly designed molecular tools are expected to facilitate the exploration and quantification of those species in different aquatic habitats where they exist, adding a major contribution to future efforts for exploiting them as resources for blue biotechnology.