This work considers the effects of using the chitosan/sodium dodecyl sulfate (SDS) pretreated filters (CSPFs) as the antiseptic filters on the bioaerosol penetration. Three concentrations of chitosan solutions and chitosan/SDS mixing solutions were used to pretreat the polypropylene fibrous filter to make them antiseptic. The λ virus bioaerosol was generated using a Collison nebulizer, as the challenged bioaerosol. The effects of various factors, including the face velocity and the relative humidity on the bioaerosol collection characteristics were evaluated. Experimental results suggested the pretreatment of chitosan/SDS did have an antiseptic effect on virus bioaerosol and increase the inactivation mechanism. Experimental findings also revealed that the addition pretreatment of SDS did raise the inactivation effect of the chitosan. When adding SDS in the chitosan solution, the ammonium groups could appear and present the antiseptic benefit more obviously. Moreover, the antiseptic of the pretreated filter decreased with face velocity and RH.