Aquatic ecosystems in urban areas are often ecologically impaired but causative factors are rarely identified, thus proving a challenge for environmental management agencies to improve ecosystem health. Causal effects may emerge by considering multiple lines of evidence at different levels of biological organisation to investigate impairment. This study was undertaken in the Maribyrnong River in the north-west of Melbourne, Victoria. Numerous pollution sources enter the catchment including runoff from residential properties, industrial estates and wet weather sewage discharges. The aims of this study were to identify the primary factors causing biological impact in the Maribyrnong River and to determine if biota are showing signs of exposure to sewage–related contaminants. To elucidate biological impairment we conducted chemical (pesticides, metals, nutrients, hydrocarbons) and faunal and flora assessments, including caging studies of mudsnails (Potamopyrgus antipodarum) and amphipods (Austrochiltonia subtenuis), oxidative stress biomarkers in shrimp (Parataya australiensis) and flatheaded gudgeon (Philypnodon grandiceps), endocrine disruption-related endpoints in fish (P. grandiceps; Gambusia holbrooki) and toxicological studies with algae (Scenedesmus sp), chironomids (Chironomus tepperi) and amphipods. Numerous metals, hydrocarbons and pesticides, including zinc, nickel, copper, lead, organochlorine and synthetic pyrethroid pesticides, were detected throughout the catchment at levels exceeding the ANZECC guidelines. These contaminants were determined to be originating from two main sources. Pollution is likely to be affecting aquatic fauna, with P. antipodarum, A. subtenuis and C. tepperi populations ecologically impaired. The usefulness of considering multiple lines of evidence approach for aquatic biomonitoring and integration into management practices to achieve successful remediation will be discussed.