Milk thistle with its active ingredient silymarin, is effective in treating various liver diseases and has antimicrobial properties. An experiment aimed at study of the chemical compositions and antimicrobial activity of alcoholic extract of milk thistle seed on 8 species of gram-positive bacteria, Staphylococcus aureus, Bacillus cereus, gram-negative bacteria, Salmonella enterica, Proteus mirabilis, Escherichia coli (E.coli), Shigella Dysenteria, Klebsiella Pneumonia and Pseudomonas aeruginosa. by using disk diffusion and macrodilution methods was conducted based on a completely randomized design in the veterinary laboratory of Miyaneh Branch of Islamic Azad University in 2017. The composition of methanolic extract of milk thistle seeds were evaluated using HPLC and the presence of two substances, silybin A and B, were identified as the main constituents of silymarin. In the antibacterial test, methanolic extract of milk thistle seeds was effective against Staphylococcus aureus, Bacillus cereus, Salmonella enterica, Proteus mirabilis and Escherichia coli but had less inhibitory effect compared to Gentamicin and Ampicillin. In disk diffusion, Staphylococcus aureus had the highest inhibitory zone and the highest MIC and MBC. In Bacillus cereus and Salmonella enterica, the growth inhibition zone diameter was approximately equal to that of Staphylococcus, and Proteus and Escherichia coli showed a smaller diameter zone than Staphylococcus. It seems that milk thistle methanolic extract contains antiseptic substances with antibacterial effects. Therefore, it is recommended to study other strains.