Even though sediment Escherichia coli is a common source of contamination in freshwater environments, their characteristics such as virulence genes, biofilm forming capacity, antibiotic resistance, and level of reactive oxygen species (ROS) are not well understood. This study examined E. coli from freshwater sediments, sewage, and a collection of O157 isolates. Multiplex PCR analysis revealed that none of the sediment and 3% of the sewage isolates contained diarrheagenic E. coli genes. However, 12.5% of sediment and 42.4% of sewage isolates contained one or more uropathogenic genes. Biofilm assays determined that sediment E. coli were significantly better biofilm formers (p < 0.001), forming 2- and 3.5-times as much biofilm as sewage and O157 E. coli, respectively. The antibiotic-resistance patterns illustrated that the E. coli belonged to three distinct groups with sediment being most susceptible and sewage being most resistant (p < 0.05). Although the planktonic cellular ROS levels of the three sample groups tested were not different from each other, the level of ROS in biofilm cells was eightfold lower than their planktonic counterparts (p < 0.001). Cells with higher ROS tolerance have been shown to have more tolerance to bactericidal antibiotics; however, the antibiotic-resistance levels of the bacteria in this study did not correlate to their cellular ROS concentrations.