In this research work, Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites have been synthesized via an eco-friendly green synthesis technique, solgel route and co-precipitation method respectively for the assessment of antibacterial activity as well as in vivo toxicity. The XRD patterns confirm the formation of Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites showing monoclinic structures. Crystallite size and lattice strain are calculated by Scherrer equation, Scherrer plot and Willimson Hall plot methods. Average crystallite size measured for Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites by Scherrer equation, Scherrer plot and WH-plot methods are (5.1, 13.9, 11.5)nm, (5.4, 14.2, 11.3)nm and (5.2, 13.5, 12.0)nm respectively. Optical properties such as absorption peaks and band-gap energies are studied by UV–vis spectroscopy. The FTIR peaks at 513 cm−1, 553 cm−1and 855 cm−1confirms the successful synthesis of Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites. The antibacterial activity of synthesized Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposites is examined against two gram-negative (Escherichia coliand pseudomonas) as well as gram-positive bacteria (Bacillus cereusandStaphylococcus aureus) at dose 25 mg/kg and 40 mg/kg by disk diffusion technique. Zone of inhibition for Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO at dose 40 mg/kg againstE. coli(gram − ve) are 12 mm, 17 mm and 18 mm respectively and againstPseudomonas(gram − ve) are 28 mm, 19 mm and 21 mm respectively. While the zone of inhibition for Bi2O3/GO and Bi2O3/CuO/GO at dose 40 mg/kg againstB. cereus(gram + ve) are 8 mm and 8.5 mm respectively and againstS. aureus(gram + ve) are 5 mm and 10.5 mm respectively. These amazing results reveal that Bi2O3, Bi2O3/GO and Bi2O3/CuO/GO nanocomposite as a kind of antibacterial content, have enormous potential for biomedical applications. In addition, the in vivo toxicity of synthesized Bi2O3/CuO/GO nanocomposite is investigated onSwiss Albinomice at dose of 20 mg/kg by evaluating immune response, hematology and biochemistry at the time period of 2, 7, 14 and 30 days. No severe damage is observed in mice during whole treatment. Thepvalue calculated by statistical analysis of hematological and biochemistry tests is nonsignificant which ensures that synthesized nanocomposites are safe and non-toxic as they do not affect mice significantly. This study proves that Bi2O3/CuO/GO nanocomposites are biocompatible and can be explored further for different biomedical applications.