In this research, 36 cows with clinical mastitis were treated with the Chinese herb injection Ruyuankang which was developed by our group. Milk samples of pre and post treatment of Ruyuankang were collected for isolation and characterization of pathogenic bacteria. The dynamic changes of pathogenic bacteria were investigated as well. The results indicated that Ruyuankang could effectively inhibit the common pathogenic bacteria of mastitis which result in the negative rate of pathogenic bacteria 71.74%.