Mycobacterium tuberculosis (MTB) is transmitted through the air, and can be captured on ventilation air filters. People handling these filters may be exposed to infectious material. We studied the survival of strains of Mycobacterium on high efficiency particulate air (HEPA) filter material. We used a model ventilation system to evaluate survival over time of Mycobacterium chelonae and H37Ra (an avirulent stain of MTB) aerosolized and then captured on HEPA filter material. Survival curves for M. chelonae incubated at 55% and 75% RH under static conditions were not different with less than 4% survival at 24 hours. H37Ra was subjected to continuous airflow at the design airflow for the filter material following deposition on the HEPA filter material, and less than 0.1% of cells survived to 48 hours (RH not controlled). H37Ra was resistant to immobilized biocide (trimethoxysilylpropyl dimethyloctadecyl ammonium chloride) on HEPA filter material as well as the same biocide in solution. Finally, survival of H37Ra and virulent MTB strain (H37Rv) were not different following deposition onto HEPA filter material from liquid suspension and incubation under static conditions.