The rapid rise in natural gas extraction utilizing hydraulic fracturing increases the potential for contamination of surface and ground water from chemicals used throughout the process. Hundreds of products containing more than 750 chemicals and components are potentially used throughout the extraction process, including over one hundred known or suspected endocrine disrupting chemicals. We hypothesized that a selected subset of chemicals used in natural gas drilling operations and also surface and ground water samples collected in a drilling-dense region of Garfield County, CO would exhibit estrogen and androgen receptor activities. Water samples were collected, solid-phase extracted, and measured for estrogen and androgen receptor activities using reporter gene assays in human cell lines. Of the 39 unique water samples, 89%, 41%, 12%, and 46% exhibited estrogenic, anti-estrogenic, androgenic, and anti-androgenic activities, respectively. Testing of a subset of natural gas drilling chemicals revealed novel anti-estrogenic, novel anti-androgenic, and limited estrogenic activities. The Colorado River, the drainage basin for this region, exhibited moderate levels of estrogenic, anti-estrogenic, and anti-androgenic activities, suggesting that higher localized activity at sites with known natural gas related spills surrounding the river might be contributing to the multiple receptor activities observed in this water source. The majority of water samples collected from sites in a drilling-dense region of Colorado exhibited more estrogenic, anti-estrogenic, or anti-androgenic activities than reference sites with limited nearby drilling operations. Our data suggest that natural gas drilling operations may result in elevated EDC activity in surface and ground water.