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 ofS. 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.