The optimization of culture conditions for the production of bioactive exopolysaccharide (EPS) by Ganoderma gibbosum, employing response surface methodology has been carried out in the present study. Initially, Plackett–Burman design was applied to identify different factors affecting the EPS yield and among these factors FeSO₄, almond oil and glucose were found to be the most significant. The interaction between these factors was analyzed using a central composite design which predicted the optimum values for FeSO₄, almond oil, and glucose to be 0.635 g/L, 2.286%, and 30.05 g/L, respectively. The determined (R²) value was 0.9848 which indicated that the model was in a good agreement with the experimental results. In order to validate the conditions subsequent verification experiment was also carried out, as there was no significant difference between the predicted value (3.36 g/L) and actual value (3.41 g/L), the model was considered to be a good fit for EPS production. The EPS produced was further evaluated for its anti-oxidant potential. The in-vitro results suggested that the EPS exhibited remarkable scavenging properties on 2, 2-diphenylpicrylhydrazyl (DPPH) and 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) ions at a dosage of 1 mg/mL suggesting potent anti-oxidant activity.