Photolysis of nitrate, a prevalent constituent in agriculturally impacted waters, may influence pathogen attenuation in such systems through production of hydroxyl radical (•OH). This study focuses on the efficacy of •OH generated during nitrate photolysis in promoting E. coli die-off as a function of extracellular polymeric substances (EPS) coverage. EPS levels of four E. coli isolates were systematically altered through a sonication extraction method and photochemical batch experiments with a solar simulator examined isolate viability loss as a function of time in nitrate solutions. E. coli viability loss over time exhibited two regimes: an initial induction time, ts, with little decay was followed by rapid exponential decay characterized by a first-order disinfection rate constant, k. Increasing steady-state •OH concentrations enhanced E. coli viability loss, increasing values of k and decreasing ts values, both of which were quantified with a multitarget bacterial disinfection model. Notably, at a given steady-state •OH concentration, values of ts and k were independent of EPS levels, nor did they vary among the different E. coli strains considered. Results herein show that while •OH generated via nitrate photolysis enhances rates of disinfection in surface water, the mechanism by which •OH kills E. coli is relatively insensitive to common bacterial variables.