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Bioaerosol contamination problems have led to the need for new technologies that effectively collect and inactivate airborne microorganisms. Typical nanomaterial‐based filter membranes are usually sterilized using photocatalysts, electrical stimulation, and thermal treatment, which are expensive and require additional devices and cumbersome manufacturing. In this study, a membrane with nanotopographical features is manufactured via a catalyst droplet‐based procedure to mechanically damage airborne bacteria. The catalyst droplets are used as templates for in situ novel puncturable nanopillar growth on the membrane surface. Numerical simulations and microscopic observations show that puncturable nanopillars with a thin and rough nano‐edge are advantageous for rupturing the bacterial cell compared to flat nanopillars without a thin edge. A puncturable nanostructured air filter (PNAF) is compared to a bare air filter and exhibits higher bioaerosol collection efficiencies (>98% and 89.3–95.7%, respectively). PNAF is tested under breathing conditions as part of a face mask, where it effectively captures and deactivates E. coli aerosols through a mechano‐bactericidal effect, resulting in the inhibition of bacterial proliferation and finally death. Thus, PNAF can be applied as an air purifier or face mask filter for bioaerosol collection presenting antibacterial effects without external stimulation. [ABSTRACT FROM AUTHOR]