Miswak (Salvadora persica L.) root extract (SPE) is known for its high antimicrobial and antioxidant activities. In this study, antimicrobial nanocomposites were prepared based on carboxymethyl cellulose (CMC), cellulose nano-fiber (CNF) and SPE. Scanning electron microscopy (SEM) was used distribution of CNF in CMC matrix and energy-dispersive X-ray (EDX) spectroscopy proved the existence of minerals and sulfur-containing compounds in SEP-activated nanocomposite. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy were used for CNF and SPE effects on structure of resulted nanocomposite. Incorporation of CNF increased the ultimate tensile strength (UTS) and decreased the elongation at break point (EB) while adding of 200 and 400 mg/ml caused increase of EB without decrease of UTS. Thermal gravimetric analysis (TGA) showed that SPE improved the heat stability of the nanocomposites. The pure SPE and SPE-activated nanocomposites showed good antibacterial effect against both Staphylococcus aureus (gram positive) and Escherichia coli (gram negative) bacteria.