Contamination of surface waters with microbial pollutants from fecal sources is a significant human health issue. Identification of relative fecal inputs from the mosaic of potential sources common in rural watersheds is essential to effectively develop and deploy mitigation strategies. We conducted a cross-sectional longitudinal survey of fecal indicator bacteria (FIB) concentrations associated with extensive livestock grazing, recreation, and rural residences in three rural, mountainous watersheds in California, USA during critical summer flow conditions. Overall, we found that 86% to 87% of 77 stream sample sites across the study area were below contemporary Escherichia coli-based microbial water quality standards. FIB concentrations were lowest at recreation sites, followed closely by extensive livestock grazing sites. Elevated concentrations and exceedance of water quality standards were highest at sites associated with rural residences, and at intermittently flowing stream sites. Compared to national and state recommended E. coli-based water quality standards, antiquated rural regional policies based on fecal coliform concentrations overestimated potential fecal contamination by as much as four orders of magnitude in this landscape, hindering the identification of the most likely fecal sources and thus the efficient targeting of mitigation practices to address them.