To assess the effectiveness of iodine-treated biocidal filter media against bacterial spore aerosols. Bacillus subtilis spores were aerosolized and introduced into a filtration system. Both treated and untreated filters exhibited high viable removal efficiency (>99.996%) with negligible variation in pressure drop during the entire experiment. The viability of collected spores on the filter was investigated by enumeration of spores extracted from the filter by vortexing. At room temperature and low relative humidity (RH), the survival fraction of the treated filter was significantly lower than that of the untreated filter (P-value < 0.05). Meanwhile, at room temperature and high RH and at high temperature and high RH, the survival fractions on the treated medium were statistically the same as the untreated control at room temperature and low RH. Both treated and untreated filters achieved excellent viable removal efficiency for spores. The pressure drop of the treated filter was not affected by the iodine treatment. The viability of collected bacterial spores was decreased because of the exertion of iodine disinfectant. The evaluation demonstrates that the iodine-treated filter is a viable medium for respiratory protection against infectious spore aerosols. The results warrant further evaluation of smaller biological agents, which exhibit higher penetration.