Use of an air filter material combined with antibacterial agents is one of the most effective methods to resolve the problem of air filter contaminated by pathogenic microbes. ε-Polylysine and Natamycin are two biogenic antimicrobials that have been widely applied in recent years because of their high antibacterial efficiency, harmlessness to human body and environmental friendliness. In this paper, a novel antibacterial air filter material was prepared by immobilizing ε-Polylysine and Natamycin onto fiberglass high efficiency air filter media by acrylate adhesive bonding. The mechanical properties, aerosol filtration properties, and antibacterial properties were then evaluated. An improvement in the mechanical properties of the material prepared was seen compared to the untreated filter media. The filtration efficiency of the material prepared for particle aerosols and bioaerosols both greater than 99.997%. Antibacterial efficiency of the material prepared against Staphylococcus aureus and Escherichia coli in suspensions were both greater than 99.99% compared to the untreated filter media. The anti-mildew effect against Aspergillus niger in suspension was strong compared to the untreated filter media. Antibacterial efficiency of the material prepared against bacteria in bioaerosols was greater than 99.99%. Observed with Scanning Electron Microscope, most bacteria on antibacterial filter media appeared to be dead. Thus, antibacterial air filter material prepared by immobilizing bio-antimicrobials on fiberglass had a strong inhibitory effect against gram-positive bacteria, gram-negative bacteria and fungi, with no impairment of the intrinsic properties. This kind of material appears to be promising for application in air cleaning and biological protection fields.