Tuberculosis (TB) is considered as one of the most fatal infectious diseases nowadays. Several traditional anti-tuberculosis drugs like isoniazid have been largely applied; however, they are associated with toxicity and poor anti-TB treatment. So, the fabrication of new alternative anti-TB drugs containing natural biopolymers for TB treatment has attracted great attention in recent years because of their remarkable features: biodegradability, biocompatibility and non-toxicity. Therefore, their medicine is very effective with low side effects compared with synthetic drugs. Our current work intends to engineer chitosan biguanidine (ChBG) nanoparticles as a new safe and high-efficient anti-TB drug using one-pot, green, cost-effective ionic gelation method. The chemical structure of as-formed materials was chemically confirmed using various analysis techniques: H-NMR, FTIR, SEM, and TEM. TEM results have proved the formation of uniformly well-distributed ChBG nanoparticles with a small particle size of similar to 38 nm. The inhibitory activity of these prepared nanoparticles was investigated against the growth of three different M. tuberculosis pathogens such as sensitive, MDR, and XDR, and in a comparison with the isoniazid drug as a standard anti-tuberculosis drug. The antituberculosis assay results showed that ChBG NPs attained MIC values of 0.48, 3.9, 7.81 mu g/mL for inhibiting the growth of sensitive, MDR, and XDR M. tuberculosis pathogens compared to bare Ch NPs (15.63, 62.5 > 125 mu g/mL) and the isoniazid drug (0.24, 0, 0 mu g/mL), respectively. Moreover, cytotoxicity of the ChBG NPs was examined against normal lung cell lines (Wi38) and was found to have cell viability of 100 % with the concentration range of 0.48-7.81 mu g/mL.