The current study investigates the anticancer effects of PEGylated chitosan nanoparticles (CS NPs) coloaded with betaine (BT) and nedaplatin (ND) on breast adenocarcinoma (MCF-7) cells and breast cancer-bearing rats. Hereof, the ionotropic gelation approach was implemented for the synthesis of PEG-uncoated and PEG-coated CS NPs encompassing either BT, ND, or both (BT-ND). The sizes of the developed BT/CS NPs, ND/CS NPs, and BT-ND/CS NPs were 176.84 ± 7.45, 204.1 ± 13.6, and 201.1 ± 23.35 nm, respectively. Meanwhile, the sizes of the synthesized BT/PEG-CS NPs, ND/PEG-CS NPs, and BT-ND/PEG-CS NPs were 165.1 ± 32.40, 148.2 ± 20.98, and 143.7 ± 7.72 nm, respectively. The surface charges of the fabricated nanoparticles were considerably high. All of the synthesized nanoparticles displayed a spherical form and significant entrapment efficiency. Release experiments demonstrated that the PEGylated and non-PEGylated CS NPs could discharge their contents into the tumor cells' microenvironments (pH 5.5). In addition, the NPs demonstrated an outstanding ability to reduce the viability of the MCF-7 cell line. In addition, BT-ND/PEG-CS NPs were found to be the strongest among all NP preparations, where they caused around 90% decrease in the size of mammary gland tumors in rats compared to vehicle-treated animals.