Cancer constitutes a large group of diseases and is a prime cause of death worldwide. Erhlich ascites carcinoma (EAC), an extemporaneous murine adenocarcinoma, is one of the well-recognized models in cancer research. Diosgenin (DGN) is a naturally occurring steroidal saponin from fenugreek and wild yam. Polymer nanoparticles have potential applications in biomedicine, but one of the important concerns is their safety. The present study aimed to evaluate the anti-cancer activity of diosgenin-encapsulated PLGA nanoparticles (PLGA-DGN NPs) against Ehrlich ascites carcinoma. The synthesized PLGA-DGN NPs were characterized by dynamic light scattering and surface zeta potential measurement, UV-vis spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The antineoplastic potency of PLGA-DGN NPs against EAC cells was assessed through cell viability assay, oxidative stress, chromatin condensation, and cell cycle arrest by flow cytometry. The cytotoxicity of PLGA-DGN NPs in EAC cells indicates the MTT assay revealed that high cytotoxic efficacy of PLGA-DGN NPs against EAC cells with the IC50 value of PLGA-DGN NPs was 7.91µg/ml. The oxidized glutathione (GSSG) increased compared to EAC control and decreased the level of reduced glutathione (GSH) in PLGA-DGN NPs, DGN rather than EAC control. In EAC cells, ROS formation was determined by H2DCF2DA staining using fluorescence microscopy showed PLGA-DGN NPs elevated the intracellular ROS generation at their respective IC50 doses indicating potent apoptotic activity compared to DGN-treated EAC cells and EAC control. PLGA-DGN NPs exhibited cytotoxicity and induced apoptosis by increasing reactive oxygen species, chromatin condensation, cell cycle arrest at the G2/M phase, expression of pro-apoptotic proteins, and mitochondrial dysfunction in EAC. All the results indicate that PLGA-DGN NPs exert cytotoxic, oxidative stress, and apoptotic effect. In conclusion, the findings demonstrate that PLGA-DGN NPs may act as potential beneficial agents in carcinoma chemoprevention