Fatty acids, organic solvents and surfactants were investigated for their stimulatory effects on the growth of fungal mycelium and production of exopolysaccharide (EPS) by submerged fermentation of an edible mushroom Pleurotus tuber-regium. Addition of 3.0 g/L Tween 80 at the late stage of exponential growth phase provided the best stimulatory effect on mycelial biomass and fungal EPS production with a 51.3 and 41.8% increase, respectively. The chemical structure of the EPS produced with addition of Tween 80 was found to be a glucomannan with similar monosaccharide composition and glycosidic linkages but a significantly lower molecular weight (3.18 ± 0.09 × 10(6) Da), compared to the control (4.30 ± 0.12 × 10(6) Da). These two EPS could significantly inhibit the growth of chronic myelogenous leukemia K562 cells in a dose dependent manner, with an estimated IC(50) value of 43.7 and 47.6 μg/mL, respectively. The use of stimulatory agents to enhance production of bioactive fungal EPS can be applied in other fungal fermentation processes for enhancing production of useful metabolites.