Understanding the transport behavior, survival, and persistence of pathogens such as Escherichia coli O157:H7 in the subsurface is essential to protection of public health. In this study, the transport of E. coli O157:H7 in a two-dimensional bench-scale sand aquifer system, hereafter referred to as the sandbox, was investigated, with a focus on the impact of biofilm development on E. coli retention and survival. Biofilm growth was initiated through flushing with unsterilized groundwater and addition of glucose, nitrate, and phosphate. Retention of E. coli from an injection test in clean sand, prior to promotion of biofilm growth, was approximately 9%. Subsequent to biofilm growth, 47% of injected E. coli cells were retained under similar flow conditions. After 10 d of no flow, sterile water was flushed through the biofouled sandbox and substantial concentrations (up to 1.5 × 105 cells/mL) of E. coli were measured in the effluent indicating that E. coli had survived the starvation period. Confocal laser scanning microscopy revealed that E. coli were located not only on the surface but also within the biofilm. Imposition of starvation conditions resulted in biofilm sloughing and possible mobilization of biofilm-associated E. coli.