Nanotechnology has become an extensive area of study due to the peculiar properties of nanoparticles. Chitosan is considered the most promising material for future applications. The purpose of this study was to highlight the antimicrobial and hepatoprotective properties of Chitosan nanoparticles (CTS), as well as their efficacy against multidrug-resistant pathogens and various applications as a natural antioxidant in the biomedical field. CTS were prepared with or without surfactant (L—lecithin, Tween 80) based on inotropic gelation of chitosan with sodium alginate. The nanoparticle obtained displayed a spherical shape with a particle size ranging from 54.3±20.8 to 1256±16.8 nm, zeta potential ranging from 24±1.2 to 30.8±1.1 mV, and polydispersity index ranging from 0.274±0.09 to 0.553±0.06. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) calculations were used to evaluate the antibacterial activity of CTS against four human pathogens: Bacillus subtilis ATCC6633, Staphylococcus aureus NRRLB-767, Escherichia coli ATCC25955, and Pseudomonas aeruginosa ATCC101455. The MIC values were 156.3, 39.4, 78.1, and 78.1 ug/mL, while the MBC values were 500,156.3, 312.5, and 312.5 ug/mL, respectively. S.aureus was the most susceptible, while B. subtilis was the most resistant. The hepatoprotective effect was determined by measuring antioxidant, antiapoptotic, and inflammatory biomarkers, histopathological and immunohistochemical studies. Hepatoprotective results showed a remarkable ameliorative effect against hepatotoxicity induced by CCl4.