The mycoparasite Ampelomyces quisqualis strain ITA3 is a biocontrol agent of powdery mildew disease but the mycelium and spore production of the fungus still needs to be developed. The objective of this research is to find out the optimum conditions for high mycelium and spores production of the strain ITA3. At submerged-fermentation stage (vegetative growth), the nutrition factors (carbon source, nitrogen source and C/N ratio) were optimized whereas at solid surface-fermentation stage (sporulation) culture nutrient medium was optimized for lower cost. The results showed that, out of three kinds of carbon source, sucrose (jaggery) gave the highest mycelia growth compared to dextrose and glucose. The organic nitrogen sources (i.e. potato extract, yeast extract, malt extract, urea) gave a better significant growth for A. quisqualis strain ITA3 (P < 0.001) compared with the inorganic nitrogen source (sodium nitrate, ammonium chloride). The C/N ratio and agitation speed were also optimized for higher mycelia growth of the fungus. It was observed that the harvested mycelia from stationary growth phase of ITA3 (6 days) transferred into a solid potato extract agar plate gave higher sporulation (4 days) in comparison to transferring it into solid jaggery and potato dextrose agar plates. At this optimum condition the strain ITA3 produced an average of 5 x 108 spores per g of mycelium after 96 hours. These outcomes indicate that the higher mycelium and spore production of A. quisqualis strain ITA3 can be achieved with a two-step fermentation system by the short period with cheaper culture medium