Fermenting conditions for antibacterial activity substance produced by Streptomyces TD-1 was optimized by orthogonal and homogeneous test design method. The effects of different experimental factors on Streptomyces TD-1 producing antibiotic substance were studied. The results showed that the most important factors which influence the antibacterial activity were glucose, soy flour, FeSO4, K2HPO4 and KNO3; the optimized cultural conditions were the initial pH 7.15, culture temperature 30℃, 6% of inoculum, the amount of shake flask 90mL/250mL, shaking speed 180r/min and fermentation time 8d. The final results showed that the antifungal activity of the fermentation broth could increase by 53.38%.