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Introduction Novel approaches for the synthesis of gold nanoparticles (AuNPs) are of great importance due to its vast spectrum of applications in diverse fields, including medical diagnostics and therapeutics. Te presented study reports the successful AuNPs’ synthesis using <i>Artocarpus heterophyllus</i> Lam. extract, and provides detailed characterization and evaluation of its antibacterial potential. [b]Objective[/b]. The aim was to develop a cost-effective and environmentally friendly synthesis method of gold nanoparticles using aqueous fruit extract of<i> Artocarpus heterophyllus</i> Lam. as a reducing and capping agent, which has proven activity against human pathogens, such as microbial species [i]E.coli[/i] and <i>Streptobacillu</i> sps. Material and Methods Characterizations were carried out using ultraviolet-visible (UV-Vis) spectrophotometry, scanning electron microscopy (SEM), energy dispersive X-ray and Fourier-Transform infra-red spectroscopy (FT-IR). Results SEM images showed the formation of gold nanoparticles with an average size of 20–25 nm. Spectra collected while infra-red analysis contained broad peaks in ranges from 4000–400 cm^-1 . Conclusions It can be concluded that the fruit of <i>Artocarpus heterophyllus</i> Lam. can be good source for synthesis of gold nanoparticles which showed antimicrobial activity against investigated microbes, in particul <i>E. coli</i>, and <i>Streptobacillus</i>. An important outcome of this study will be the development of value-added products from the medicinal plant <i>Artocarpus heterophyllus</i> Lam. for the biomedical and nanotechnology-based industries.