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During animal waste agricultural applications, the major concern is pathogen spreading, which may contaminate surface water and groundwater. Among the pathogenic microorganisms found in animal waste, Salmonella typhimurium and Escherichia coli O157:H7 are of particular concern. When transported in sub-surface agricultural soil, S. typhimurium and E. coli O157:H7 are captured at the air–water–sediment interfaces through physical interactions. Because in situ colloids contribute to the formation of air–water–sediment, their mobilization affects the transport of S. typhimurium and E. coli O157:H7. The impact of irrigation rates on in situ colloid mobilization and S. typhimurium and E. coli O157:H7 transport was investigated in intact soil columns collected from an agricultural site in Gadsden County of Florida, USA. The columns were irrigated with sterilized nano-pure deionized water to mobilize the colloids in the soil by stepwise increases in flow rate. For each flow rate, after colloids were mobilized and steady state was reached, S. typhimurium and E. coli O157:H7 were introduced. The cumulative amount of released in situ colloids increased linearly with the irrigation rates (R2 = 0.986–0.996) and transport of the bacteria was enhanced after colloid mobilization. Interactions of the bacteria with the sediments and the air-water interface were characterized: these played an important role in controlling S. typhimurium and E. coli O157:H7 retention in soil.