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The present study quantifies the transport of Escherichia coli pathogenic O157:H7 and nonpathogenic K12 strains in water-saturated Quincy sand (QS) columns amended with oxidized (OX) or unoxidized (UO) pine wood (PW) or pine bark (PB) biochar produced at either 350 or 600 °C. Our results showed that (1) the addition of oxidized biochar into QS columns enhanced the transport of E. coli O157:H7 by 3.1 fold compared to the unoxidized counterparts, likely because of an increase in the repulsive forces due to their higher negative charge densities. (2) The retention of E. coli O157:H7 was 3.3 fold higher than that of E. coli K12 in all biochar-amended sand columns. (3) Increased application rates of unoxidized PW600 biochar from 0 to 20 wt % led to a reduction in the transport of E. coli O157:H7 and K12 from 98 to 10% and from 95 to 70%, respectively. Our data showed that mixing sand with PW350-UO at a 20 wt % application rate almost completely retained the pathogenic E. coli in the subsurface, suggesting that utilizing sand mixed with biochar can act as a promising biofilter capable of protecting natural aquafers from pathogens.