Withania somnifera (Ashwagandha) is a member of the Solanaceae family, which is famous for its phyto-pharmacological effects, such as being anti-inflammatory, antioxidant, anti-stress, immunomodulatory, and anticancer. The study sought to develop and assess chitosan-coated PLGA nanoparticles containing ashwagandha extract. The nanoparticle formulation technique was contemplated to address the diverse challenges linked to ashwagandhas, including intestinal absorption, rapid medication release, and bioavailability concerns. We used the Box-Behnken design in Design-Expert Software to find out how the independent variables PLGA, chitosan concentration, and sonication time affected the size of the CS-PLGA NPs. PDI and the efficacy of trapping. The improved formulation had a particle size of 187.1 nm, a PDI of 0.148, and a zeta potential of 31.3 mV. Optical microscopy and SEM indicate that the particles exhibited a smooth, spherical morphology and homogeneous dimensions. The improved formulation has an entrapment efficiency of 84.28%. The in vitro drug release investigation indicates that the enteric coating of the CS-PLGA NPs formulation inhibited drug release in simulated gastric fluid (SGF) and demonstrated prolonged drug release compared to pure ashwagandha, with drug release kinetics conforming to the Korsmeyer-Peppas model. Moreover, the CS-PLGA NPs were assessed for in vitro antioxidant activity using DPPH and for in vitro cytotoxicity employing CaCo-2 cell lines. The findings indicated superior antioxidant activity compared to pure ashwagandha and improved cytotoxicity against CaCO-2 cells. The investigations found that CS-PLGA NPs released drugs over a lengthy period of time. The mixture has strong antioxidant properties and worked better on cancer cells.