Abstract: The mycoparasite Ampelomyces quisqualis strain ITA3 is a biocontrol agent ofpowdery mildew disease but the mycelium and conidia production of the fungus still needs to bedeveloped. The objective of this research is to find out the optimum conditions for highmycelium and conidia production of the strain ITA3. At submerged-fermentation stage(vegetative growth), the nutrition factors (carbon source, nitrogen source and C/N ratio) wereoptimized whereas at solid surface-fermentation stage (sporulation) culture nutrient medium wasoptimized 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 organicnitrogen sources (i.e. potato extract, yeast extract, malt extract, urea) gave a better significantgrowth 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 forhigher mycelium growth of the fungus. It was observed that the harvested mycelium fromstationary growth phase of ITA3 (6 days) transferred into a solid potato extract agar plate gavehigher sporulation (4 days) in comparison to transferring it into solid jaggery and potato dextroseagar plates. At this optimum condition the strain ITA3 produced an average of 5 x 108 conidia perg of mycelium after 96 hours. These outcomes indicate that the higher mycelium and sporeproduction of A. quisqualis strain ITA3 can be achieved with a two-step fermentation system bythe short period with cheaper culture medium.