We assessed the prevalence of verotoxigenic Escherichia coli (VTEC) in chamois (Rupicapra rupicapra) and livestock grazing on a mountain pasture in Austria during June–August 2009. We detected VTEC throughout the sampling period in high numbers in cattle as well as in chamois, leading to the assumption that the degree of contamination of the environment is high. This is the first report of pathogenic E. coli identified in chamois, implicating chamois as a new potential reservoir of these zoonotic pathogens. Because the study area also serves recreational purposes, there is a risk of humans acquiring infection via direct or indirect contact.