Due to the wide and inappropriate use of the antibiotics, the development of new resistant strains of bacteria to the most of common antibiotics has become a serious problem in public health; so there is a strong stimulant to continuity developing new and effective antimicrobial agents. Nanotechnology considers as a magic tool to explore and treat the difficult problems of medical sciences. The confluence of nanotechnology and microbiology solves several biomedical problems, and also can revolutionize the health and medicine fields. Recently reports prove that carbon-based nanomaterials like multiwalled carbon nanotubes (MWCNTs) show potent antimicrobial properties. The nanocomposite chitosan-multiwalled carbon nanotube (Ch/MWCNT) was synthesized by the process of sonication for 20 min and examined their antibacterial activity. Seven different concentrations of MWCNT were used in the preparation of Ch/MWCNT (from 5 to 100 mg). The prepared differently concentrated MWCNT nanocomposite was characterized using TEM and FTIR. According to TEM results which showed that the morphology of MWCNTs were obtained in the form of small tubes of a length. FTIR show that the presence of the C=C absorption at the wave number 2344 cm^-1which confirm the successful incorporation between chitosan and MWCNT. For antimicrobial activity estimation, the serial dilution method was used towards Gram positive bacteria (Staphylococcus aureus NRC 23516, Methicillin-resistant Staphylococcus aureus (MRSA) NRC 629012) and Gram negative bacteria (Escherichia coli 0157H7 and Pseudomonas aeruginosa ATCC10145). The composite Ch/MWCNT showed higher antimicrobial activity against both Gram negative and Gram positive bacteria; P. aeruginosa, Staphylococcus aureus; respectively with increasing MWCNT concentrationtill ratio 1:4. The nanocomposites are highly differentiable at the low concentration; 1% concentration of the multifunctional nanocomposite is very effective against the tested microbes. Minimal inhibitory concentrations (MIC) of Ch/MWCNT (100:50 Ch/MWCNT) against Gram negative and Gram positive pathogenic bacteria between 0.5 and 0.0625 μg, and the growth inhibition effect was observed in a concentration-dependent species.