Abstract The effect of two commonly used fertilizers, DAP (diammonium phosphate) and urea was studied on the freshwater flagellate Euglena gracilis using the automatic biotest ECOTOX. NOEC and EC 50 values for various parameters like motility, velocity, cell shape and gravitaxis were calculated. The NOEC and EC 50 values obtained for DAP were much lower than those for urea; i.e. DAP showed a stronger inhibitory effect as compared to urea. The inhibition caused by DAP increased with increasing exposure time over 24 h but urea showed no augmentation with increasing exposure time. Application of DAP resulted in an increased pH and high concentrations of ammonia but urea did neither affect the pH nor affect the ammonia concentration. Recovery experiments in deionized water after urea application showed a reconstitution of motility after 72 h. After an application of 1.35 g L −1 (24 h EC 50 for motility) DAP motility recovered after 72 h but motility did not recover when the concentration was doubled (2.7 g L −1). The EC 50 values obtained were compared with the EC 50/LC 50 values reported for other aquatic organisms and were found to be comparable with the reported values.