The present study describes the Facial preparation of bacterial-resistant cellulose acetate (CA) membranes by using novel non-toxic antibacterial cyanoacetyl microcrystalline cellulose (NACAMC). Different concentrations of (NACAMC) were blended with CA to prepare three antibacterial cellulosic membranes namely: M1, M2 and M3 having hindrance activity against tested strains. The Fourier-transformed infrared spectroscopy (FT-IR) was used to investigate the changes in chemical and physical membrane characteristics. The obtained membranes (M1, M2, and M3) were visually evaluated by scanning electron microscopy (SEM). M2 and M3are characterized by a pore-free upper surface and a porous bottom surface of the membranes in comparison with the CA blank membrane. The water uptake ratio was measured to be 175%, 426%and 707% for M1, M2and M3 respectively. Also, the average pore size distribution for prepared membranes was measured. The Mechanical properties of M1 and M2 were measured which indicates that the tensile strength and elongation are higher in M2 than M1. Antibacterial activity for membranes and (NACAMC) against 5 pathogenic strains were evaluated. These results could be developed for several uses as in wound dressing applications, food packaging, and water treatment antifouling membranes.