Green synthesis of metal oxide nanoparticles (MO NPs) offers a sustainable approach for developing biocompatible anticancer agents. In this study, α-Fe?O? and ZnO nanoparticles (NPs) were synthesized using an aqueous extract of Salvadora persica (miswak) twigs as a natural reducing and stabilizing medium. Comprehensive physicochemical characterization confirmed the formation of polycrystalline, spherical, and monodispersed NPs, with average sizes of ~24 nm for α-Fe?O? and ~21 nm for ZnO NPs. Both nanomaterials demonstrated significant antimicrobial activity against Staphylococcus aureus. However, ZnO NPs exhibited superior biological performance, including higher antioxidant capacity (82.15%) and greater cytotoxic potency (LC??: 0.81 µg/mL) compared to α-Fe?O? NPs. Hemocompatibility assays revealed minimal hemolytic activity, indicating good compatibility of ZnO NPs with human red blood cells. Zeta potential measurements (−39 mV for α-Fe?O? and −41.3 mV for ZnO NPs) reflected excellent colloidal stability. In vitro anticancer evaluation showed that miswak-derived ZnO NPs exerted stronger antiproliferative effects than α-Fe?O? NPs against colorectal (HT-29) and hepatocellular (HepG2) carcinoma cell lines, with IC?? values of 54.71 µg/mL and 29.89 µg/mL, respectively. Flow cytometric analysis confirmed enhanced induction of both early and late apoptosis in tumor cells treated with ZnO NPs. Furthermore, in vivo studies using a chronic myeloid leukemia (CML) rat model demonstrated that ZnO NPs were more effective in normalizing white blood cell counts and reducing disease-associated weight loss. Histopathological analysis of spleen tissues further supported the superior therapeutic efficacy of ZnO NPs. Nevertheless, further investigation is required to ensure safety, reproducibility, scalability, and cost-effective production for translational application. The integration of traditional medicinal knowledge with modern nanocarrier systems, highlight the potential of Salvadora persica-mediated ZnO NPs as a promising, biocompatible nanotherapeutic candidate for cancer management.