<p>Plant-mediated green synthesis has emerged as a sustainable and cost-effective strategy for the production of gold nanoparticles (AuNPs). In this study, gold nanoparticles were synthesized using an aqueous extract of <i>Virola oleifera</i> resin (AuNPs-Vo), and their synthesis parameters were optimized through factorial experimental design, followed by physicochemical characterization and cytotoxicity assessment. Factorial experimental designs were applied to identify optimal conditions for obtaining stable, concentrated, and monodisperse nanoparticles, evaluating extract concentration, pH, temperature, reaction time, and agitation. The plant extract was characterized by High-Performance Liquid Chromatography coupled with Ultraviolet–Visible Detection (HPLC-UV/Vis) and Liquid Chromatography–Mass Spectrometry (LC-MS), while the nanoparticles were analyzed by UV-Vis (Ultraviolet–Visible Spectroscopy), Transmission Electron Microscopy (TEM), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), Dynamic Light Scattering (DLS), zeta potential, and Raman spectroscopy. Nanoparticle stability was evaluated through flocculation assays under different pH, temperature, and ionic strength values. Cytotoxicity was assessed in Vero cells employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays after exposure to three AuNPs-Vo concentrations and their precursors (extract and HAuCl₄), along with the analysis of oxidative stress biomarkers: Reduced Glutathione (GSH), Glutathione S-transferase (GST), Hydrogen Peroxide (H₂O₂), Catalase (CAT), Malondialdehyde (MDA), Protein Carbonyl (PTC or Carb) and Total Antioxidant Capacity (CAOT). The most influential synthesis parameters were pH, reaction time, and extract concentration, with optimal conditions (about concentration and size) at pH 8.3, 6 minutes, and 1.0 mg mL⁻¹, respectively. Polyphenols, polyols, and flavones were identified as key agents for the reduction and stabilization of Au³⁺. The AuNPs-Vo presented an average gold concentration of 21 mg L⁻¹, predominantly spherical morphology, particle sizes of 15–21 nm, negative surface charge, and high stability at pH 3–9. Cytotoxicity assays demonstrated high cell viability (&gt; 85%) and no significant induction of oxidative stress (p &gt; 0.05), indicating low toxicity compared to precursor reagents. In some exposures to AuNPs-Vo, there was a significant decrease in carbonylated proteins (approximately 50%). These findings highlight the potential of phytochemicals derived from Virola oleifera as sustainable reducing agents for the production of stable compounds with promising applications.</p>