Recent focus on the preparation of antimicrobial surfaces using cellulose nanofibers (CNF) has gained considerable attention. In this work, functionalization of CNF films in 100% aqueous solution with three different aminosilanes, including 3-aminopropyl trimethoxysilane (APMS), 2-aminoethyl 3-aminopropyl trimethoxysilane (DAMS)and 3-2-(2-aminoethylamino) ethylamino propyl-trimethoxysilane (TAMS) is reported for the fabrication of contact active antimicrobial materials. Grafted CNF films were comprehensively characterized by FTIR, TGA, contact angle, elemental analysis, solid-state 29Si NMR, FEG-SEM and SEM-EDX. It was found that all the silanes were grafted on the surface of nanofibers without any change in the morphology or fibril structure through different grafting efficiency, depending on the aminoalkyl chain length. The effect of variable aminoalkyl length and initial grafting concentration was analyzed against gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) by qualitative and quantitative standards. The most promising results were obtained with 3-2-(2-aminoethylamino) ethylamino propyl-trimethoxysilane at very low concentration which completely restrict bacterial growth after 24 h with Gram-positive bacteria. This study, for the first time, established the relationship between the aminoalkyl chain length and its corresponding antibacterial activity.