Technical Abstract: When bacterial pathogens from livestock contaminate drinking water supplies, they can cause outbreaks of different forms of gastroenteritis. Of the different pathways by which bacterial pathogens can enter drinking water, one has received little attention to date; that is, because soils are often considered to be near perfect filters, and the transport of bacterial pathogens through the subsoil to groundwater has received very little attention. The objective of this study was to examine the concentrations of fecal bacterial composition in aquifer material impacted by contaminants from different sources. The concentrations of different bacterial species such as heterotrophic bacteria, Escherichia coli, and Enterococcus were determined after running tertiary treated water through two tanks containing aquifer material. Enterococcus faecalis primers targeting the ddl gene which encodes the D-Ala : D-Ala ligase and produces a 941 DNA base pair product upon amplification and primers for E. faecium which produced a 658 DNA base pair product were used to identify the two species in the samples. A PCR assay based on the partial sequence of the '-D-glucoronidase gene (uidA) for specific detection and differentiation of E. coli populations was used to confirm the presence of E. coli after biochemical test. The biochemical test overestimated the percentage of E. faecium in our samples, but the PCR assay with the ddl gene produced 100% specificity with Enterococcus faecalis. The biochemical test was 91.5% specific in identifying E. coli. To resolve the discrepancy between the biochemical test results and the PCR confirmation, the 16S rRNA genes of questionable isolates were sequenced. Biochemical test for identification of Enterococcus faecalis agreed with PCR assay, but discrepancies were noted for Enterococcus faecium and E. coli. Therefore, direct identification of Enterococcus faecium and E. coli from environmental samples may provide a serious challenge for accurate identification of fecal bacteria contamination of drinking water sources.