Concentrations of human health-related microorganisms in runoff from agricultural plots (0.75 m × 2 m) treated with fresh and aged cattle manure, swine slurry and no manure (control) were determined. Three consecutive simulated rainfall events, producing 35 mm rainfall and separated by 24 h, were carried out for each plot. Fecal indicator (Escherichia coli, enterococci, Clostridium perfringens and coliphage) loads released in rainfall runoff from plots treated with fresh cattle manure, aged cattle manure and swine slurry treatments ranged from 5.52 × 105 to 4.36 × 109, 3.92 × 104 to 4.86 × 108, and 9.63 × 105 to 3.05 × 108, respectively. Plot runoff concentrations of protozoa (Cryptosporidium oocysts and Giardia cysts) ranged from 1.65 × 105 to 1.04 × 106, 2.93 × 103 to 2.75 × 105, and 9.12 × 104 to 3.58 × 106 for fresh cattle manure, aged cattle manure and swine slurry plot treatments, respectively. These results suggest that large microbial loads could be released via heavy precipitation events that produce runoff from livestock manure-applied agricultural fields, of even modest size, and could have a significant impact on water bodies within the watershed. Because of the lack of multiplication in the environment, highly elevated concentrations in manured land runoff, and correlation to protozoan parasite presence, Clostridium may be an alternative indicator for livestock manure contamination.