The John Muir Trail (JMT) in the Sierra Nevada Mountains of California is one of the most popular alpine wilderness trails in the United States, where backpackers depend on trailside water sources for more than 335 km (208 miles). This study addressed the risk of acquiring waterborne disease by analyzing prevalence and changes in coliform bacteria and Escherichia coli (E. coli) in lakes and streams adjacent to the central JMT. Chlorophyll-a levels were also measured as an indicator of high elevation eutrophication. Categories of environmental land use which might affect water quality were defined as: Pristine areas rarely traversed by humans; Backpack off-trail areas not traversed by pack or stock animals; and Multiuse areas with backpacker and animal use. We analyzed surface water at 36 different sites three separate times over an eight week period in the summer of 2008. Chlorophyll-a concentration increased significantly in Backpack and Multiuse sites over the summer months, but not in Pristine sites. Similar results were obtained for coliforms, with prevalence also increasing significantly over the summer months in Backpack and Multiuse sites. There was a much higher prevalence of E. coli in Multiuse sites compared to Pristine and Backpack sites. Our study provides evidence pack and stock animals serve as a source of microbial contamination of water along this section of trail.