Metal and metal oxide nanoparticles have gained huge interest due to their unique chemical and physical attributes that confer antimicrobial potential against a wide spectrum of pathogenic bacterial strains. Hence, the objective of the present study focused on synthesis of simple, cost-effective and environmentally friendly biofunctionalized cuprous oxide nanoparticles using root extract of Withania somnifera as antibacterial agents. The average size of the nanoparticles was found to be 410 nm with an energy band gap of 4.63 eV as confirmed by scanning electron microscopy and UV–visible spectroscopy, respectively. The chemical composition of the biofunctionalized Cu2O NPs was confirmed with the help of energy-dispersive X-ray analysis (EDAX). Vibrational studies of the cuprous oxide nanoparticles by Fourier transform infrared spectroscopy (FT-IR) gave a distinctive absorption band at 626 cm−1. The bactericidal effects of synthesized nanoparticles are attributed to different bioactive compounds, such as phthalic acid, purin-6-amine, hexadecanoic acid and benzene dicarboxylic acid, as analysed by gas chromatography–mass spectrometry (GC–MS). Finally, the antibacterial activities of cuprous oxide nanoparticles on Bacillus, Enterobacter, Escherichia and Mycobacterium were investigated by calculating the minimum bactericidal concentration (MBC) and colony forming unit (CFU) content by the broth dilution method. The biofunctionalized Cu2O NPs appeared to have strong antibacterial activity with 100% of inhibition against Escherichia coli, Enterobacter aerogenes and Mycobacterium smegmatis at MBC value 0.8 mg/ml whereas 100% inhibition against Bacillus subtilis at MBC value 1.0 mg/ml. The findings of our study suggest that the biofunctionalized Cu2O NPs coated with bioactive compounds from root extracts of Withania somnifera could be a promising antimicrobial agent for a wide range of pathogenic strains, especially Mycobacterium tuberculosis.