Microbial contamination of surface waters, a substantial public health concern throughout the world, is typically identified by fecal indicator bacteria such as E. coli. Thus, monitoring E. coli concentrations is critical to evaluate current conditions, determine restoration effectiveness, and inform model development and calibration. An often overlooked component of these monitoring and modeling activities is understanding the inherent random and systematic uncertainty present in measured data. In this research, a review and subsequent analysis was performed to identify, document, and analyze measurement uncertainty of E. coli data collected in stream flow and stormwater runoff as individual discrete samples or throughout a single runoff event. Data on the uncertainty contributed by sample collection, sample preservation/storage, and laboratory analysis in measured E. coli concentrations were compiled and analyzed, and differences in sampling method and data quality scenarios were compared. The analysis showed that: 1) manual integrated sampling produced the lowest random and systematic uncertainty in individual samples, but automated sampling typically produced the lowest uncertainty when sampling throughout runoff events; 2) sample collection procedures often contributed the highest amount of uncertainty, although laboratory analysis introduced substantial random uncertainty and preservation/storage introduced substantial systematic uncertainty under some scenarios; and 3) the uncertainty in measured E. coli concentrations was greater than that of sediment and nutrients, but the difference was not as great as may be assumed. This comprehensive analysis of uncertainty in E. coli concentrations measured in streamflow and runoff should provide valuable insight for designing E. coli monitoring projects, reducing uncertainty in quality assurance efforts, regulatory and policy decision making, and fate and transport modeling.