A technique is presented that can be used to estimate the changes in physical structure in a natural biofilter packing medium, such as compost, over time. The technique applies information from tracer studies, grain size distribution, and pressure drop analysis to a model that estimates the number of channels, average channel diameter, number of particles, and specific surface area of the medium. Important operational factors, such as moisture content, pressure drop, and sulfate accumulation also were evaluated both in a conventionally operated biofilter and in one operated with periodic compost mixing. In the conventionally operated laboratory-scale compost biofilter, hydrogen sulfide (H2S) removal efficiency decreased from 100% to approximately 90% over 206 days of operation. In a similar system, operated with compost mixing, the H2S removal efficiency was maintained near 100%. Variations in media moisture conditions and specific surface area can explain the results observed in this study. Under conventional operation, drying near the inlet disintegrated the compost particles, producing a large number of particles and flow channels and increasing the specific surface area. At the top of the column, where moisture was added, particle size increased and specific surface area decreased. In the column with media mixing, moisture content, particle size, and specific surface area remained homogeneous.