The effects of biochar feedstock, pyrolysis temperature, and application rate (1 and 2%) on the transport of two Escherichia coli isolates through a fine sand soil under water-saturated and partially saturated conditions were investigated in column experiments. Biochars from two feedstocks (poultry litter and pine chips) and pyrolyzed at two temperatures (350 and 700 °C) were evaluated. Both biochars pyrolyzed at 700 °C resulted in significant reductions in E. coli transport, with greater reductions observed with the pine chip biochars. For the low temperature biochars, increased transport was observed for the poultry litter biochar whereas reduced transport was observed for the pine chip biochar. In general, the effect of biochar application on E. coli transport was more pronounced in the unsaturated soils and for the 2% application rates. Large differences were also observed between the two isolates indicating that bacterial surface properties play a role in how biochar affects E. coli transport.