Bench testing of petroleum biodegradation rates in vadose zone soils is typically associated with errors because transport conditions in laboratory systems are different from those found in the vadose zone. This work addressed the effect of soil structure and gas transport properties on hydrocarbon biodegradation in the unsaturated zone and we present a novel method for measuring biodegradation rates in intact and undisturbed soil columns of 100 cm3. To determine whether soil structure and gas diffusivity, defined as the ratio of the gas diffusion coefficient in soil to that in free air (Dp/D0), influence the outcome of aerobic benzene biodegradation experiments, measurements using identical sandy soils were performed on (i) undisturbed 100-cm3 core samples; (ii) sieved (2-mm) and repacked 100-cm3 core samples; and (iii) soil samples (10 g) prepared as slurry microcosms. While slurry reactor experiments changed the first-order rate constant (kw,1) significantly compared with undisturbed core samples, this was not the case for soil cores that had been sieved and repacked. This suggests that soil structure on a millimeter scale does not affect aerobic biodegradation in relatively unstructured sandy soils. Within differently textured soil cores, the biodegradation rate was found to increase with gas diffusivity when Dp/D0 < 0.02. This establishes gaseous O2 and petroleum vapor diffusion and distribution in soil profiles as a controlling factor for natural biodegradation of petroleum vapors.