This study was based on the hypothesis that groundwater-derived biofilms may provide a reservoir for coliform or pathogenic bacteria as has been observed in drinking water distribution systems. Escherichia coli, labelled with green fluorescent protein, was found to colonize all layers of mixed-population biofilms developed in association with indigenous groundwater micro-organisms in a laboratory-scale reactor. Biofilm-associated E. coli was removed at a slower rate from the reactor flasks than planktonic E. coli under a continuous flow regime. During flow-through of groundwater, planktonic E. coli removal was slower in flasks containing coverslips for enhanced biofilm development compared to a control flask without coverslips. Conversely, during flow-through of treated effluent, planktonic E. coli removal was faster in flasks with coverslips compared to without. Removal of attached E. coli was also fastest in the coverslip-containing flasks with effluent flow-through. This suggests that an increase in available nutrients may reduce E. coli survival potential due to either enhanced competition for nutrients or enhanced antagonism by the indigenous microbial population. Under identical conditions, GFP-labelled Pseudomonas aeruginosa was found to persist in the biofilms for longer than E. coli, most notably when exposed to flow-through of treated effluent. However, prolonged persistence of P. aeruginosa in the effluent could not be attributed to an association with the biofilms. This study has shown that under certain conditions the presence of mixed-population biofilms may limit the survival potential of enteric bacterial pathogens introduced into groundwater.