Aims: Isolate and characterize water enterococci from the South Nation River drainage basin, an area dominated by agriculture. Methods and Results: A total of 1558 enterococci were isolated from 204 water samples from the South Nation River obtained over a 3-year period. PCR was used to identify isolates to the species level and characterize them for carriage of 12 virulence determinants. Antibiotic resistance was evaluated phenotypically. Enterococcus faecalis (36.4%), Enterococcus faecium (9.3%) and Enterococcus durans (8.5%) were the major enterococci species isolated. Enterococci carrying more than two virulence determinants were more frequently detected in the summer (59.6%) than in other seasons (<= 37.6%). Very few isolates (<= 2.0%) were resistant to category I antibiotics ciprofloxacin and vancomycin. Conclusions: Comparison of major water enterococci species with major faecal enterococci species obtained from various host groups (human, domesticated mammals and birds, wildlife) in this drainage basin suggest that water enterococci may have varied faecal origins. The low level of antibiotic resistance among enterococci suggests that dispersion of antibiotic resistance via water-borne enterococci in this watershed is not significant. Significance and Impact of the Study: The data obtained in this study suggests that water enterococci in the SNR have a faecal origin and that their potential impact on public health regarding antibiotic resistance and virulence determinants is minimal.