Cancer remains a major global health worry, particularly in developing nations, where economic limitations necessitate affordable therapeutic alternatives. The Siddha medicine, a traditional South Indian system of medicine, incorporates several formulations with potential anticancer properties, including Thirunethira Chooranam (TNC). To evaluate the in vitro antioxidant and cytotoxic, and in vivo anticancer activity of TNC using the Dalton’s lymphoma ascites (DLA) cells-induced tumor model in Swiss albino mice. Antioxidant activity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Cytotoxic potential was examined against estrogen-responsive breast cancer cell line (MCF-7), lung adenocarcinoma epithelial cell line (A549), hepatocellular carcinoma cell line (HEP-G2), human cervical cancer cell line (HeLa), and African green monkey kidney (Vero) cell line via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In vivo efficacy was evaluated in DLA-bearing mice by analyzing hematological, biochemical, cancer cell count, survival, and histopathological parameters. TNC demonstrated dose-dependent antioxidant activity (half-maximal inhibitory concentration [IC₅₀] = 7 mg/mL). The MTT assay revealed selective cytotoxicity, with the strongest effect against MCF-7 cells (IC₅₀ = 14.39 mg/mL) while sparing normal Vero cells. In vivo studies showed significant restoration of hematological and biochemical indices, an 80% increase in life span, reduction in tumor cell count, and partial restoration of hepatic and renal architecture. The results validated that TNC demonstrated its in vitro antioxidant and cytotoxic effects, and in vivo anticancer efficacy as a cost-effective and potential drug for cancer treatment.