Aquatic ecosystems in mountainous regions are crucial for fulfilling natural and anthropogenic water demands around the world. This study integrates Escherichia coli (E. coli) enumeration, microbial source tracking (MST), and environmental DNA (eDNA) analysis to identify sources of fecal contamination in oligotrophic mountain waters. Conducted in an area with intense tourism and traditional reindeer herding, this research addresses the urgent need to identify fecal pollution sources to safeguard the water quality of these vital ecosystems. Our study reveals that E. coli levels vary significantly across different locations and times, suggesting varied sources of contamination from humans, wildlife, and livestock animals. MST techniques, alongside eDNA analysis, provided insights into the complex patterns of fecal pollution, allowing for the distinction between human and animal contributions to water contamination. Our findings highlight the importance of combining various analytical methods to track fecal pollution sources effectively, and to develop targeted strategies for water quality management.