Runoff from open-lot animal feeding areas contains microorganisms that may adversely affect human and animal health if not properly managed. One alternative to full manure containment systems is a vegetative treatment system (VTS) that collects runoff in a sediment basin and then applies it to a perennial vegetation (grass) treatment area that is harvested for hay. Little is known regarding the efficacy of large-scale commercial VTSs for the removal of microbial contaminants. In this study, an active, pump-based VTS designed and built for a 1200-head beef cattle feedlot operation was examined to determine the effects of repeated feedlot runoff application on fecal indicator microorganisms and pathogens over short-term (2 wk) and long-term (3 yr) operations and whether fecal bacteria were infiltrating into deeper soils within the treatment area. In a short-term study, fecal bacteria and pathogen numbers declined over time in soil. Measurements of total coliforms and Enterococcus counts taken on control soils were not effective as fecal indicators. The repeated application of manure-impacted runoff as irrigation water did not enrich the pathogens or fecal indicators in the soil, and no evidence was seen to indicate that pathogens were moving into the deeper soil at this site. These results indicate that large-scale, active VTSs reduce the potential for environmental contamination by manure-associated bacteria. Also, this study has implications to full-containment systems that apply runoff water to land application areas (cropland) and the fate of pathogens in the soils of land application sites.