Human sewage contamination of waterways is a major issue in the United States and throughout the world. Models were developed for estimation of two human-associated fecal-indicator and three general fecal-indicator bacteria (HIB and FIB) using in situ optical field-sensor data for estimating concentrations and loads of HIB and FIB and the extent of sewage contamination in the Menomonee River in Milwaukee, Wisconsin. Three commercially available optical sensor platforms were installed into an unfiltered custom-designed flow-through system along with a refrigerated automatic sampler at the Menomonee River sampling location. Ten-minute optical sensor measurements were made from November 2017 to December 2018 along with the collection of 153 flow-weighted discrete water samples (samples) for HIB, FIB, dissolved organic carbon (DOC), and optical properties of water. Of those 153 samples, 119 samples were from event-runoff periods, and 34 were collected during low-flow periods. Of the 119 event-runoff samples, 43 samples were from event-runoff combined sewer overflow (CSO) influenced periods (event-CSO periods). Models included optical sensor measurements as explanatory variables with a seasonal variable as an interaction term. In some cases, separate models for event-CSO periods and non CSO-periods generally improved model performance, as compared to using all the data combined for estimates of FIB and HIB. Therefore, the CSO and non-CSO models were used in final estimations for CSO and non-CSO time periods, respectively. Estimated continuous concentrations for all bacteria markers varied over six orders of magnitude during the study period. The greatest concentrations, loads, and proportion of sewage contamination occurred during event-runoff and event-CSO periods. Comparison to water quality standards and microbial risk assessment benchmarks indicated that estimated bacteria levels exceeded recreational water quality criteria between 34 and 96% of the entire monitoring period, highlighting the benefits of high-frequency monitoring compared to traditional grab sample collection. The application of optical sensors for estimation of HIB and FIB markers provided a thorough assessment of bacterial presence and human health risk in the Menomonee River.