The aims of nano oncology are to detect, target and treat cancer cells without any side effects. The present study describes the microbial synthesis of biocompatible nanoparticles of silver (AgNPs), gold (AuNPs) and their alloy (Ag/AuNPs) for hepatoprotective activity against diethylnitrosamine (DEN)-induced liver cancer in a Sprague Dawley (SD) rat model. The crystalline nature and physicochemical features of the nanoparticles were identified by Fourier transform infra-red (FT-IR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and selected area electron diffraction (SAED) analysis. Based on the instrumental analysis, the synthesised nanomaterials were found to be spherical in shape and have an average size in the nano region. Nitrate reductase was characterized after partial purification of the culture filtrate via polyacrylamide gel electrophoresis and its molecular weight was determined as ∼45 kDa. Furthermore, the IC₅₀ values of the AgNPs, AuNPs and Ag/AuNPs on HepG2 cells were determined as 38.42 μg ml⁻¹, 43.25 μg ml⁻¹ and 39.20 μg ml⁻¹, respectively, and the antioxidant potential of the nanoparticles was also systematically analyzed. The No-Observed-Adverse-Effect-Level (NOAEL) for the AgNPs was determined to be 2000 mg per kg of body weight (bw) from an acute toxicity test. Similarly, the NOAEL of AuNPs and Ag/AuNPs were calculated as 1000 mg per kg bw. Based on the in vivo studies, a significant tumour reduction (∼45 to 65%) was observed in the nanoparticle-treated animals, which was further confirmed by hematological, biochemical, TEM and histopathological analysis. Immunohistochemistry analysis confirmed the presence of BAX antibodies, up to immunoreactive (3+) level in treated animals. These results strongly suggest the potential anticancer activities of AgNPs, AuNPs and Ag/AuNPs against DEN-induced liver cancer and they could be potential candidates for effective nano drug development.