Soil aggregates are an important structural component of the soil matrix that could harbor Escherichia coli and provide an environment for its survival and water flow reentering. Knowledge of the exact pore locations within soil aggregates obtained using X-ray computed microtomography opens new opportunities for understanding microorganism movement within the soil matrix. The first objective of this study was to assess E. coli spatial distribution within soil macroaggregates and its potential for leaving the aggregates with the saturated water flow. The second objective was to study the relationships between the distribution and movement of E. coli within soil aggregates and the aggregates’ internal pore structures. We studied aggregates from the top (A) horizon of conventionally tilled (CT) and no-till (NT) corn–soybean–wheat rotations and native succession vegetation (NS) treatments at NSF Long-Term Ecological Research site, southwest Michigan. The results confirmed that E. coli movement in soil aggregates was mainly driven by water flow via capillary forces. E. coli redistribution was most pronounced in CT aggregates, followed by NT, and was almost negligible in NS aggregates. Pore characteristics that positively contributed to E. coli redistribution through the aggregates were the maximum flow in the aggregate centers and the ratio of the maximum flow and pore tortuosity. The E. coli retention in the aggregate’s centers was positively related to porosity, percent of medium and large pores, and pore tortuosity.