Monitoring recreational waters for fecal contamination by standard methodologies involves culturing indicator bacteria, such as fecal coliforms and enterococci. Delayed reporting of microbial water quality parameters increases the likelihood of public exposure to pathogens of fecal origin, making the development of rapid methods important for public health protection. A rapid assay for enterococci was developed using a combined ultrafiltration-biosensor procedure. Twelve 100-liter water samples were collected from upper Tampa Bay over a 9-month period. The samples were collected on site by dead-end hollow-fiber ultrafiltration. Postfiltration processing of the initial retentates included sonication and micrometer-level sieve passage to remove interfering particles. Centrifugation was utilized for secondary concentration. Grab samples were collected simultaneously with the ultrafiltered samples. Concentrations of enterococci in all grab and ultrafiltration samples were determined by the standard method (EPA method 1600) for calculation of recovery efficiencies and concentration factors. Levels of enterococci increased twofold in initial retentates and by 4 orders of magnitude in final retentates over ambient concentrations. An aliquot of each final retentate was adsorbed onto polystyrene waveguides for immunoassay analysis of enterococci with a microfluidic fiber optic biosensor, the Raptor. Enterococci were detected when concentrations in the ambient water exceeded the regulatory standard for a single sample (105 CFU/100 ml). The combined ultrafiltration-biosensor procedure required 2.5 h for detection compared to 24 for the standard method. This study demonstrated that enterococci can be detected rapidly using on-site ultrafiltration, secondary concentration, and biosensor analysis.