A series of novel fluorescent compounds were used to stain protozoans in pure culture and in complex environmental biofilm communities. Confocal laser scanning microscopy was used to image fluor stained, NiSO4 immobilized, living protozoans in situ. Most of the stains, specific for eucaryotic cell structures, such as Paclitaxel-BodipyFL, CellTracker, DiOC(6)(3), LysoTracker Red, MitoFluor Green and Rhodamine 6G showed potential for in situ staining of protozoa. However, some of the stains also stained bacterial or polymeric biofilm constituents. Nevertheless after staining, the usually transparent protozoan cells became clearly visible within the complex architecture of environmental biofilms. With respect to staining protozoan cells only and differentiation from other biofilm constituents, CellTracker showed the highest specificity. By using this approach, protozoa can be identified and distinguished within a complex habitat by virtue of (1) specific stains targeted for eucaryotic cell features, especially CellTracker, and (2) staining in combination with information on their size and morphology. With respect to fluorochrome concentration the approach has to be fine-tuned according to the specific characteristics of the sample as well as the identity and physiological status of the protozoa.