To investigate the antimicrobial activities of the Streptommyces galilaeus AF1 metabolites on Streptococcus agalactiae,a spectrophotometer and counting plate colony was used to detect the effect of the metabolites against S. agalactiae and a comparative test on the efficacy with certain antimicrobial agents was conducted. The results showed that the metabolites ofS. galilaeus AF1 have strong effects against S. agalactiae and the minimal inhibitory concentration was 38 μg/mL with the does-time-effect relationship. The scanning electron microscopy results indicated that the cell walls of S. agalactiae were ruptured severely after being treated with the metabolites. The 96 h LC50 value for zebrafish were 27.44 mg/L and 35.44 mg/L by static method and feeding method,respectively and the acute toxicity was low. This study showed that the metabolites of S. galilaeus AF1 have good antibacterial activity against S. agalactiae and provides a new clue for prevention and control of S. agalactiae.