During the last decades, the Rosetta branch of the River Nile received a great number of drainage wastes (domestic, agricultural, and industrial) that affected different aquatic organism's life. Accordingly, characterizing the biological aspects of this water body is necessary. The current study aims to survey the biodiversity of flora and fauna along the Rosetta branch to assess the environmental status of this area. Eight sites were selected along the Rosetta branch during 2018 for samples collection. Results indicated the presence of seven macrophyte species, dominated by the free-floating and emergent species Eichhornia crassipes and Echinochloa stagnina, respectively. Bacillariophyceae had the highest number of species compared with other groups of attached algae and occupied the first and the greatest predominance position. Epiphytic microinvertebrates associated with the floating plant Eichhornia crassipes recorded the highest numbers of species and groups during summer and the diversity was affected by the heavy load of pollution discharged to the Nile (especially at El-Rahawy region). Five groups of zooplankton (Rotifera, Copepoda, Cladocera, Meroplankton, and Protozoa) were documented and identified based on their morphological characters. High numbers of bacterial indicators of pollution exceeding the permissible limits were obtained, inferring the harmful effects of drains discharge on the water quality at the Rosetta branch. Physico-chemical parameters showed a great correlation between different biological aspects. The current study confirmed the effects of environmental factors on surveyed aquatic organisms, which could be used as a guide for evaluating water quality in the evaluated area.