Intestinal Escherichia coli caused diarrhea in chicken makes serious damage directly to the chicken culture industry. Bacteriophage therapy is able to control the diarrhea in chickens effectively. In this study, the biosafety of bacteriophages was evaluated for treating intestinal pathogenic E. coli, which induced diarrhea in chickens. Ten bacteriophages were isolated from feces of chickens with diarrhea using the ill-chicken intestinal pathogenic E. coli 3-2 as target organism. Three bacteriophages propagated on E. coli 3-2 with relative big and clear plaques were selected and used together for toxicity experiment and evaluating the effect of therapy on chicken weight gain. In 3 weeks of trial, no mice given with or without mixed bacteriophages died, and the weight of mice of the experimental group did not show significant difference to the control group after 3 weeks infection. Besides remarkable decreasing the death rate of chickens with diarrhea, treatment of mixed bacteriophages also promoted the weight gain and saved the diet consumption as the utilize rate of diet increased 11% compared with the control group. These observations indicated that a mixture of three bacteriophages would be biosafe for rapid and effective preventing pathogenic E. coli infections.