The main object of this research is chemical modification of the nanofibrillated cellulose (NFC) surface with a phenanthridinium silane salt to develop durable non-leaching antibacterial surface. Initially, (3 trimethoxysilylpropyl) phenanthridinium iodide (TMSPhI) as an antibacterial agent was synthesized using (3-chloropropyl trimethoxysilane) (CPTMS) and phenanthridine in the presence of potassium iodide. Subsequently, NFC was cationized by reaction of its hydroxyl groups with the trimethoxysilane group of TMSPhI to prepare the modified sample (NFC-TMSPhI). The synthesized TMSPhI was characterized by FT-IR, 1H and 13C NMR spectroscopies. The modified NFC samples were also characterized by FE-SEM/EDX, XRD, TGA, elemental analysis, contact angle measurement, FT-IR, UV–Visible and fluorescence spectroscopies. The obtained NFC–TMSPhI samples presented fluorescence property at the maximum emission wavelength in the range of 539–549 nm. Additionally, the antibacterial activity of the modified samples were evaluated quantitatively against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. All the modified samples displayed promising results with at least bacteriostatic effect or bactericidal properties. Finally, the cytotoxicity of the modified sample based on normal HDFs cells was investigated that showed dose- and surface charge-dependent toxicity.