Relations between spectral absorbance and fluorescence properties of water and human-associated and fecal indicator bacteria were developed for facilitating field sensor applications to estimate wastewater contamination in waterways. Leaking wastewater conveyance infrastructure commonly contaminates receiving waters. Methods to quantify such contamination can be time consuming, expensive, and often nonspecific. Human-associated bacteria are wastewater specific but require discrete sampling and laboratory analyses, introducing latency. Human sewage has fluorescence and absorbance properties different than those of natural waters. To assist real-time field sensor development, this study investigated optical properties for use as surrogates for human-associated bacteria to estimate wastewater prevalence in environmental waters. Three spatial scales were studied: Eight watershed-scale sites, five subwatershed-scale sites, and 213 storm sewers and open channels within three small watersheds (small-scale sites) were sampled (996 total samples) for optical properties, human-associated bacteria, fecal indicator bacteria, and, for selected samples, human viruses. Regression analysis indicated that bacteria concentrations could be estimated by optical properties used in existing field sensors for watershed and subwatershed scales. Human virus occurrence increased with modeled human-associated bacteria concentration, providing confidence in these regressions as surrogates for wastewater contamination. Adequate regressions were not found for small-scale sites to reliably estimate bacteria concentrations likely due to inconsistent local sanitary sewer inputs.