We evaluated the role of soil aggregate pore size on biodegradation of essentially insoluble petroleum hydrocarbons that are biodegraded primarily at the oil-water interface. The size and spatial distribution of pores in aggregates sampled from biodegradation experiments of a clayey, aggregated, hydrocarbon-contaminated soil with relatively high bioremediation end point were characterized by image analyses of X-ray micro-CT scans and N2 adsorption. To determine the bioaccessible pore sizes, we performed separate experiments to assess the ability of hydrocarbon degrading bacteria isolated from the soil to pass through membranes with specific sized pores and to access hexadecane (model insoluble hydrocarbon). Hexadecane biodegradation occurred only when pores were 5 μm or larger, and did not occur when pores were 3 μm and smaller. In clayey aggregates, ∼ 25% of the aggregate volume was attributed to pores larger than 4 μm, which was comparable to that in aggregates from a sandy, hydrocarbon-contaminated soil (~23%) scanned for comparison. The ratio of volumes of inaccessible pores (<4 μm) to bioaccessible pores (>4 μm) in the clayey aggregates was 0.32, whereas in the sandy aggregates it was approximately 10 times lower. The role of soil microstructure on attainable bioremediation end points could be qualitatively assessed in various soils by the aggregate characterization approach outlined herein.