The optimization of microbial growth for biotechnological purposes traditionally requires an approach that uses only one variable at a time, which has many drawbacks. This research used a completely randomized approach to optimize carbon and nitrogen nutrient requirements and growth factors (pH and temperature) for Pleurotus tuber-regium in order to optimally produce biomass and extracellular polysaccharide (EPS) in shake-flask cultures. An artificial neural network (ANN) module was used to simulate the fungus-growing process and hence determine optimal conditions. The experiments demonstrated the effectiveness of the EPS fraction from P. tuber-regium in preserving hepatic cells against paracetamol-induced damage. Totals of 0.699 g biomass and 0.291 g EPS per 100 mL medium were obtained, whereas the ANN predicted 0.750 g biomass and 0.300 g EPS per 100 mL medium, thereby achieving 93.20% predictability for biomass and 73.00% predictability for EPS. Conditions for optimal EPS and biomass production for P. tuber-regium were quite different. Rat hepatic cells that had been fortified with the EPS fraction from P. tuber-regium were effectively preserved against liver damage. By using a mathematical approach, this study established optimal fermentation conditions for mycelia biomass and EPS production by P. tuber-regium and the relevant biotechnological implications.