Stormwater is a major source of Escherichia coli in urban waterways. This study assessed the chemical and bacterial contaminant load in stormwater entering an urban river using chemical analyses, E. coli enumeration and antibiotic resistance testing. Inline monitoring of five major stormwater outfalls revealed that E. coli concentrations can range from 100 to >240,000 CFU 100 ml−1 in stormwater; 15% of the samples contained >240,000 CFU 100 ml−1. E. coli levels increased from <200 CFU 100 ml−1 to between 1,500 and >20,000 CFU 100 ml−1 in the receiving waters following storm events. Metals and nutrients were significantly elevated (p < 0.05) in inline stormwater samples compared with baseflow river water. Chromium, zinc, lead and total phosphorus were co-ordinately elevated in stormwater, but E. coli levels did not correlate to this chemical signature. E. coli isolates from stormwater and river water samples (n = 2,478) were tested for resistance to 10 antibiotics and compared with over 1,800 isolates from human and non-human sources. The antibiotic resistance frequencies of isolates from human sources were considerably higher than those found in non-human sources. Isolates from human sources were resistant to multiple antibiotics; however, no specific multiple resistance pattern was found. One of five stormwater outfalls tested contained E. coli resistant to antibiotics at frequencies that were not significantly different from those of E. coli isolates from wastewater treatment plant influent (p < 0.05). The results from antibiotic resistance testing suggested that the elevated E. coli levels observed after storm events are a mixture of human and non-human sources. The results of this study demonstrate that there is a considerable E. coli load to receiving waters from stormwater and that sanitary sewage inputs may not be limited to recognized sewer overflows.