The nanostructured insect wings have inspired the development of antimicrobial surfaces with mechano-bactericidal activity. For the first time, a chevaux-de-frise-like nanostructure was fabricated through the coating of cellulose nanocrystals (CNC) onto regenerated cellulose (RC) films via vacuum filtration and the impact of contact time, temperature, and surface topography on eliminating foodborne bacteria was examined. Herein, our focus is to explore in more detail how the surface charge of CNC affects the mechano-bactericidal activity and the performance of chevaux-de-frise-like nanostructure in meat preservation. CNC with neutral (weak), negative, and positive charges were prepared by hydrochloric acid hydrolysis (HCNC), TEMPO oxidation (TCNC), and amination (ACNC), respectively, and showed similar reinforcing effects on the tensile strength (increased from 74.23 ± 1.20 to about 100 MPa) and water vapor barrier property (reduced from 1.83 ± 0.08 to about 1.20 × 10−7 g m−1 h−1 Pa−1). Among them, RC-ACNC showed the highest log reduction against Escherichia coli (0.83 ± 0.06) and Staphylococcus aureus (0.69 ± 0.04) after 5 min contact, respectively, indicating the important role of attractive force in fast eliminating bacteria upon contact. It was worth noting that, during the meat preservation test, all three CNC-coated RC films exhibited a similar 0.4 log reduction of bacteria after day 4, likely due to the same physical attachment with an extended contact time. Therefore, the construction of chevaux-de-frise nanostructure from CNC on food packaging provides a sustainable strategy to contribute to preventing bacterial growth.