In order to increase the metabolites of the strain BD3900 inhibiting the S. mutans biofilm formation, the inhibition rate of biofilm formation by Streptococcus mutans was used as indexes. The effects of the cultivation time and temperature, the carbon resources, the concentration of sucrose and the inoculum amount were investigated. The results showed that optimal cultivation conditions were composed of a cultivation period of 16 h, a cultivation temperature of 34 ℃, and a sucrose concentration of 1 g/100 mL. Under the optimized cultivation conditions, the inhibitory ability of the strain BD3900 on the formation of S. mutans biofilm was increased by 10%, compared with the results before optimization. The mechanism involved in the inhibiting of S. mutans biofilm formation by the strain BD3900 was probably decreasing the insoluble exopolysaccharides synthetization.