Nowadays, green chemistry methods are noticeable for synthesis metal nanoparticles due to affordable, easy and high reaction rate. In the present study gold nanoparticles (Au NPs) were eco-friendly synthesized using Thyme extract at the room temperature for 30 min to provide non-toxic, which can be used for different applications. Identifying properties of synthesized gold nanoparticles was done by various analytical technique including UV-Vis absorption spectroscopy approved presence of Au NPs in the solution, the functional groups of Thyme extract in the reduction and capping process of Au NPs are determined by FT-IR, Crystalline with the fcc plane approved by X-ray diffraction (XRD) pattern, energy dispersive spectroscopy (EDS) determined existence of elements in the sample, surface morphology, diverse shapes and size of present Au NPs were showed by scanning electron microscopy (SEM), atomic force microscopy (AFM) and Transmission electron microscopy (TEM). Beginning and end destroy temperature of the gold nanoparticles were determined by thermal gravimetric spectroscopy (TGA). In addition, antibacterial, antioxidant, and cytotoxicity properties of Au NPs were studied. Antibacterial activity of Au NPs was investigated on gram-positive (Bacillus) and gram-negative (Escherichia coli) by disk diffusion; also MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) were determined. DPPH free radical scavenging assay was used for antioxidant property and compared to butylated hydroxy toluene (BHT) as a standard antioxidant that showed high antioxidant activity. Synthesized Au NPs have great cell viability in a dose-depended manner and demonstrate that this method provided nontoxic for synthesis gold nanoparticles. The average diameter of synthesized Au NPs was about 35 nm.