Typical investigations into the biological consequences of suspected xenobiotics or nutrients introduced in watersheds include analytical chemistry screens of environmental samples—such as periphyton responses or studies of fish condition—which are all costly in terms of equipment, reagents, time, and human resources. An alternative is to assess pollutant effects on waterborne bacteria. A flow cytometric method was developed to yield rapid, same-day results that could be used to proactively screen for suspected chemical inputs into watersheds using water sampling methods that are identical to those in standard use. The analytical methods are microbe cultivation-independent, for use with waterborne bacteria that are typically viable but not culturable. The procedure is quick and inexpensive, generating measures of bacterial esterase that reflect metabolic activity and are sensitive and statistically robust. After phosphate-EDTA incubation to increase cell wall permeability, staining was performed with 5(6) carboxyfluorescein diacetate (enzyme activity) and propidium iodide (cell viability) with three bacterial species in exponential phase growth having been incubated with organic wastewater compounds (atrazine, pharmaceuticals [17α-ethynylestradiol and trenbolone], and antimicrobials [tylosin and butylparaben]). This method successfully detected metabolic changes in all bacterial species, with atrazine inducing the greatest change. Additional fluorescent stains can target specific microbial structures or functions of interest in a particular watershed. This biotechnology can inform analytical chemistry and study of biota at sites of interest and has the potential to be automated.