Fecal Indicator Bacteria (FIB) such as Escherichia coli (E. coli) are a leading cause of surface water impairments in the United States. However, the relative impacts of different watershed characteristics on microbial water quality in agriculturally dominated watersheds are unclear. Spatial and statistical analyses were utilized to examine relationships between watershed characteristics and FIB and a multiple regression model was created. Geometric mean E. coli concentration data were obtained for 395 ambient water quality monitoring locations in Iowa. Watersheds were delineated for thirty randomly selected monitoring locations and drainage areas ranged from 93 to 1.1 million hectares. Watershed characteristics examined include area, presence of animal units (open feed lots and confinements), percent of watershed area receiving manure application, presence of point-source discharges, and land cover. The results from the analyses reveal that the presence of animal feeding operations and agriculture, wetland, and woody vegetation land covers are the most influential watershed characteristics regarding E. coli concentration. A significant positive correlation was identified between E. coli concentration and agriculture while significant negative correlations were identified with animal feeding operations and wetland and woody vegetation. Establishing relationships between watershed characteristics and presence of E. coli is needed to identify dominant watershed characteristics contributing to pathogen water impairments and to prioritize remediation efforts.