ABSTRACT Out of a hundred horses, fecal samples Escherichia coli was isolated from 37 samples of different ages, The isolated samples had been used to examine the effect of peganum harmaline on isolated E.coli in vitro and in vivo. The goal of this research was to determine the antimicrobial activity of peganum harmaline extract by means of (ethanol: methanol 1:1) in opposition to Escherichia coli at various 40,20, 10, 5, 2,5, 1,25 and 0,625 mg/ml concentrations in each plastic and glass tube. The values of MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) for the extract against E.coli were equal to (0.625 mg/ml) for MIC and (10 mg/ml) for MBC on bacteria which cultured on glass tube while the MIC value was 40 mg/ml and MBC was10 mg/ml on plastic tube. The effect of Peganum harmaline on the formation of E.coli biofilm was investigated and the biofilm inhibitory concentrations were 40-6.25mg / ml in vitro. In vivo, a group of laboratory mice used, the LD50 of peganum harmaline extract was tested orally by up and down method and found to be 1030 mg/kg body weight, the extract used as 103 mg/kg bodyweight treatment after causing E.coli infection at 1×108 CFU/ml in laboratory mice the treated persist for two weeks in group one and three weeks in the second group as well as three week treated in the third group from the second days after infection while the control group left without treated and the mice sacrificed after second, third and fifth days of infection. The result shows histopathological changes in all treated groups, especially in the third group and that refers to the antibacterial effect of the peganum harmaline ethanolic extract when compared to the infected control group.