The present study sought to identify Escherichia coli sources in a small catchment and to use the agro-hydrological model soil and water assessment tool (SWAT) to estimate their impact on river water quality. The innovative aspects of this research are to assess the hourly variations of fecal contamination and to take these variations into account in the model to provide a better evaluation of river quality. Thus, water samples were taken weekly at the river outlet (n = 4) and 24-h monitoring sessions were performed during low and high-flow periods (n = 74). E. coli variations were found to be primarily linked to rainfall and not to resuspension mechanisms. Subdaily fluctuations and deviations were ±0.33 log10 cfu/100 mL and ±0.70 log10 cfu/100 mL for dry (<3 mm/day) and wet (>3 mm/day) weather, respectively. After river flow calibration, all known pollution sources (septic systems, manure spreading, farm discharges) were introduced into SWAT. The model reproduced the fecal contamination in the river and the use of subdaily deviations allowed us to evaluate the simulation quality and compare grab samplings with simulated daily E. coli concentration, thus confirming that the performance of the model is better when additional information on hourly concentration variations is used.