The unwavering pursuit of natural sources for sustainable synthesis of gold nanoparticles (AuNPs) is essential in nanomedicine. The present work describes the use of Decalepis hamiltonii (Wight & Arn) methanol root extracts evaluating its in vitro antioxidant assay, anti-cancer, AuNPs characterization, and antioxidant and antibacterial studies ADME/T and molecular docking. The methanol root extract has the most substantial antioxidant and enzyme properties. Using a simple reduction reaction technique, gold nanoparticles were synthesized from the roots of Decalepis hamiltonii, and their properties such as morphology, size, shape, and charge were analyzed through various methods such as UV–Vis spectroscopy, Scanning Electron Microscopy (SEM), and Dynamic Light Scattering (DLS) analysis. The synthesis of AuNPs was confirmed by a color change from yellow to purple, and biomolecules supported their reduction and stabilization according to FTIR analysis. The nanoparticles were found to have a polydispersed size, as shown by TEM analysis. This suggests a bioactive substance has good ADME/toxicity properties, indicating its potential as an anti-cancer agent. According to molecular docking studies, 1TUP could be an inhibitor with a binding energy of − 8.15 kcal/mol. The favorable ADME/toxicity properties of 1TUP suggest its potential as an anti-cancer therapeutic agent. The IC₅₀ for 2-Hydroxy-3-Methoxybenzaldehyde was confirmed to be 118.25 out of 300 g/mL, indicating its anti-cancer efficacy against MCF-7 cells. The findings of this research provide a molecular understanding of 2HMB’s anti-cancer efficacy against MCF-7 cells and support its potential use in cancer treatment. The in vitro biosynthesis and docking data indicate that 2-Hydroxy-3-Methoxybenzaldehyde is a potential biological active source and may be an excellent choice for future, significantly suitable nanomedicines.