Bottom fauna could be used as bioindicators for water quality status in various freshwater systems such as the Rosetta branch, Egypt. This branch receives several domestic, agricultural and industrial wastes. Such wastes could affect various aquatic taxa including macrobenthic invertebrates. The current investigation aimed to evaluate the water's physicochemical characteristics, microbial load and macrobenthic invertebrate biodiversity in relation to sediment analysis in the Rosetta branch, Egypt. A total of seven stations were investigated during two seasons (winter and summer) along the Rosetta branch. The sediment types in the evaluated stations were distributed as muddy gravelly sand (at EL-Rahawy and Dosouk stations), muddy sand and sandy mud (at EL-Qanater station). The water's physicochemical characteristics were evaluated. The numbers of the characterized animal species varied among the evaluated Rosetta stations. During the winter season, a total of five animal groups (Annelida, Arthropoda, Ostracoda, Mollusca and Free-living Nematodes) were detected. Concerning the second season, only three animal groups (Annelida, Arthropoda and Mollusca) were documented. The species equitability, richness and diversity index were calculated in each evaluated station. The microbial load of each sediment sample was evaluated. The fecal streptococci were high, indicating that the Rosetta branch is polluted by sewage. The results could be used for the inference of the biodiversity levels in this Egyptian ecological area. In addition, it could be used to design some informative plans for good management of biological resources in the future.