Introduction: Synthesis of gold nanoparticles (GNPs) from various biological systems have been reported, but amongst them biosynthesis of GNPs from plants is considered as the most suitable method. The aim of this study was to synthesize GNPs using chloroauric acid and hydroalcoholic stem extract (HA) of Tinospora cordifolia and to evaluate the in vitro anti-inflammatory potential of HA and the synthesised GNP followed by assessing the acute oral toxicity of the prepared herbal GNP. Materials and Methods: Anti-inflammatory activity was evaluated by using the in vitro human red blood cell membrane stabilization method and gene expression of cytokine TNF-α by isolated human peripheral blood mononuclear cells using RT-PCR. The acute toxicity test was conducted using wistar albino rats. Results: The membrane stabilizing effect demonstrated a concentration dependent anti-inflammatory activity of both HA and GNP. GNP synthesized from hydroalcoholic stem extract of T. cordifolia inhibited production of proinflammatory cytokine TNF-α by inhibiting genes expression of the cytokine. GNP was found to be non-toxic at a single dose of 2000 mg/kg with no signs of mortality during the 14 days observation period. Conclusion: The results of the said evaluation proved that formation of GNP enhanced the anti-inflammatory activity of T. cordifolia. The acute oral administration of GNP was found to be safe and not toxic. Hence, the GNP can be utilized for treatment of diseases associated with imbalanced cytokine production as in anti-inflammatory pharmaceutical formulations.