BackgroundThe forming, blending, and characterization of materials at a size of one billionth of a meter or less is referred to as nanotechnology. The objective of the current study was to synthesize ecologically friendly gold nanoparticles (AuNPs) fromGymnosporia montanaL. (G. montana) leaf extract, characterize them, assess their interaction with different types of deoxyribonucleic acid (DNA), and investigate their antioxidant and toxic capabilities.ResultsThe biosynthesized AuNPs presence was validated by a color change from yellow to reddish pink as well as using UV–visible spectrophotometer. Fourier transform infrared (FTIR) spectroscopy analysis showed the presence of phytoconstituents like, alcohols, phenols, and nitro compounds responsible for the reduction of AuNPs. Zeta sizer and zeta potential of 559.6 d. nm and − 4.5 mV, respectively, demonstrated potential stability. With an average size between 10 and 50 nm, X-ray diffraction (XRD), and high-resolution transmission electron microscope (HR-TEM), revealed the crystalline formation of AuNPs. Surface topology with 3D characterization, irregular spherical shape, and size with 6.48 nm of AuNPs was determined with the help of an atomic force microscope (AFM). AuNPs with some irregular and spherical shapes, and sizes between 2 and 20 nm, were revealed by field emission scanning electron microscope (FESEM) investigation. Shifts in the spectrum were visible when the bioavailability of AuNPs with calf-thymus DNA (CT-DNA) and Herring sperm DNA (HS-DNA) was tested. Additionally, the DNA nicking assay’s interaction with pBR322 DNA confirmed its physiochemical and antioxidant properties. The same was also found by using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which showed a 70–80% inhibition rate. Finally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay revealed that viability decreased with increasing dosage, going from 77.74 to 46.99% on MCF-7 cell line.ConclusionSynthesizing AuNPs through biogenic processes and adoptingG. montanafor the first time revealed potential DNA interaction, antioxidant, and cytotoxicity capabilities. Thus, opening new possibilities in the turf of therapeutics as well as in other areas.Graphical Abstract