Water resources situated in areas with underlying karst geology are particularly vulnerable to fecal pollution. In such vulnerable systems, microbial source tracking (MST) methods are useful tools to elucidate the pathways of both animal and human fecal pollution, leading to more accurate water use risk assessments. Here, we describe the application of a MST toolbox using both culture-dependent bacteriophage and molecular-dependent 16S rRNA assays at spring and well sites in the karstic St Imier Valley, Switzerland. Culture-dependent and molecular-dependent marker performance varied significantly, with the 16S rRNA assays displaying greater sensitivity than their phage counterpart; HF183 was the best performing human wastewater-associated marker while Rum2Bac was the best performing ruminant marker. Differences were observed in pollution regimes between the well and spring sampling sites, with the spring water being more degraded than the well site. Our results inform the choice of marker selection for MST studies and highlight differences in microbial water quality between well and spring karst sites.