Currently, the use of 'green' synthesised nanoparticles with environmentally friendly properties is considered a novel therapeutic approach in medicine. Here, the authors evaluated gold nanoparticles (AuNPs) conjugated with Tragopogon dubius leaf extract and their antibacterial activity in vitro and in vivo. Colour changes from yellow to dark brown and a peak at 560 nm on ultraviolet-visible spectroscopy confirmed the formation of nanoparticles. Additionally, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy analyses were performed to determine particle sizes and functional groups involved in gold reduction. Moreover, using standard micro-dilution and disc-diffusion assays against Klebsiella pneumoniae, Bacillus cereus, Escherichia coli, and Staphylococcus aureus, the antimicrobial properties of synthesised AuNPs were investigated. To confirm antibacterial activity, synthesised AuNPs were applied in a rat model on burn wounds infected with S. aureus, and the nanoparticles were as effective as tetracycline in bacterial reduction and wound healing. In conclusion, the synthesis of AuNPs with aqueous T. dubius extract was rapid, simple, and inexpensive, and the synthesised nanoparticles had significant antibacterial activity in vitro and in vivo.