With a focus on the impact of the Rustumiya sewage treatment facility, the physio-chemical and indicator bacterial contamination pollution of the Diyala River (Iraq) was examined. The Diyala River in Baghdad was sampled on a monthly basis from March 2022 to February 2023 for surface water and sediment. The findings demonstrated seasonal and geographical variation in physio-chemical parameters (pH, EC, salinity, and TDS) and bacteriological markers (TC, FC, E. coli, and FS). The research procedures and methods showed that some physicochemical parameters (pH, EC, TDS) in sediment exceeded their standard limits concerning aquatic life protection in spring and summer. The level of bacteriological pollution in the water and sediments, evaluated by tracking of faecal indicator bacteria (E. coli and Enterococci), during the autumn and winter seasons had the largest concentration of bacterial contaminants. In this study, the main finding was the majority of the studied stations had water classified as not recommended or unacceptable and sediment classified as acceptable or conservatively accepted. The source of contamination, determined by the ratio value of faecal coliform bacteria to faecal Streptococcus, was from human origin in the majority of studied stations, and from mixed origin during the spring and summer. Bacterial indicators in this research exhibited both quantitative and qualitative fluctuation, suggesting their potential utility as a bio-indicator for contamination of water and sediment in the Diyala River.