Agricultural landscape management and climate seasonality can influence soil structure, hydraulic conductivity, and air permeability within the context of soil water and soil gas mobility. To investigate this, in situ and laboratory-based data were collected from three agricultural landscape positions within a watershed in eastern Ontario, Canada during a growing season. Macropore classification, water infiltration tests, and air permeability measurements were conducted in situ and standard soil characterizations were carried out on soil samples. Hydraulic conductivity of the soil matrix, based on grain size data, indicated that the highest values were consistently measured in the B horizon at each landscape setting. Macropores were found to be more abundant within uncultivated drainage ditch bank soils, compared to the adjacent cropped fields. Macropores in the ditch bank soils were exclusively consisted of circular biopores, while both circular and linear macropores were observed in the cultivated field soils. Air permeability, vertical hydraulic conductivity, and horizontal hydraulic conductivity were also greater in the uncultivated soils, relative to the cultivated soils. Field saturated hydraulic conductivity measurements offered evidence of anisotropy, likely due to the vertical nature of the macropore features. Macropore disposition and extent varied over the growing season, especially in the cultivated field soils where tillage and field trafficking are physically disruptive. Seasonality of macropore development will influence temporal changes in advection-based mass exchange of gas and water in the vadose zone. Modeling of mass exchange in agricultural soils should consider time variability in macroporosity to more realistically characterize infiltration and soil gas emissions.