Improving environmentally friendly biological procedure for nanoparticles synthesis is one of the key areas of nanotechnology research. Present report focuses on, gold nanoparticles (GNPs) were synthesized at room temperature utilizing Citrus aurantifolia (C. aurantifolia) peel extract as reducing agent. The synthesized gold nanoparticles were characterized by UV– Vis spectroscopy, zeta potential (ZP), Scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). Primarily UV-Visible spectroscopy confirm the GNPs synthesis a SPR at 550 nm. The moderate stability of biosynthesized GNPs were indicated by zeta potential value which was obtainted as -17.80mv. XRD pateern showed that the crystalline nature of GNPs synthesized were of FCC nature. Various biomolecules present in Citus aurantifolia peel extract are responsible for stabilization and reduction of GNPs, presence of these biomolecules adentifies by the FTIR analysis. The biosynthesized GNPs fundamentally restrained the development of therapeutically imperative pathogenic gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). It likewise indicated significant anticancerous activity against HepG2 and A549 cell lines. The study recommends that the green synthesized gold nanoparticles could have a high potential for use in the planning of medications utilized against different ailments and were promising contender for some therapeutic applications.