Objective:Chinese Herbal Compound I were determined by screening of Radix Scutellariae,Cortex Fraxini,Radix Pulsatillae and Radix Sophorae Flavescentis.The optimal dose of Trimethoprim which combined with it against bacteria in vitro were determined.Method:The composing prescriptions were designed by L9(34) orthogonal test.The antibacterial effect of Staphylococcus aureus,Escherichia Coli and Salmonella in vitro were evaluated by double dilution method.The number of Staphylococcus aureus,Escherichia Coli and Salmonella which had been inhabited by Chinese Herbal Compound I combined with Trimethoprim for 1,2,4h and 8h,were enumerated by Plate Counts Methods.Then the killing rate of Chinese Herbal Compound I combined with Trimethoprim were calculated.The experimental data were fitted by least squares method and the optimal dose of Trimethoprim were determined.Result:The Composition of Chinese Herbal Compound I were mixed in the proportion of 1 to 4 to 1 to 2 with Radix Scutellariae,Cortex Fraxini,Radix Pulsatillae and Radix Sophorae Flavescentis.The optimal dose of Trimethoprim was 2mg/g.Conclusion:Antibacterial action and synergistic action of Chinese Herbal Compound I were significant.