The Yucatan Peninsula’s groundwater is experiencing increases in degradation due to swelling population and tourism; yet little is known about sources and transport of contaminants in drinking water supplies. The karst allows for rapid transport of microbial and chemical contaminants to the subsurface, resulting in significantly increased potential for pollution of groundwater. The objective of this research is to determine the occurrence, source, and extent of fecal contamination in the Tulum region of the Peninsula. A multi-analytical approach was undertaken in impacted and unimpacted groundwater locations; measurements included physicochemical parameters, total coliform and E. coli, Bacteroides (human vs total) and caffeine. The results indicate a variation in geochemistry from impacted to protected sites. The total coliform and E. coli show fecal contamination is wide spread. However, the presence of human Bacteriodes and caffeine in the water in the Tulum well field indicates that the recent human activities next to the well field are impacting the drinking water supply. This project is an assessment of the area’s current water quality conditions and the probable impact that the aforementioned growth would have on the area’s water supply. By applying multiple source parameter measurements, including molecular microbiology and chemical indicators it was confirmed the extent of fecal contamination of human origin covered the entire sampling region.