In order to evaluate the effect of Thymus vulgaris essential oil on isolated Lactobacillus involve with rumen acidosis, rumen fluid sample was collected from the rumen of a caw equiped with permanent fistula. The cow according to was fed by a diet containing 65% concentrate and 35% forage. Bacterial isolation was done on standard methods. Bacterial culture was done on the special media of MRS and colonies were selected and distinguished hased on their size and morphology as well as catalase, oxidase and Gram staining tests. The Thymus vulgaris essential oil was extracted by the Clevenger apparatus. The effect of Thymus vulgaris essential oils was done using the agar cup test. Thirty-six colonies of lactobacilluse genus were identified according these characteristics. It was confirmed that heterofermentative Lactobacillus with a propertion of 58% were more than the homofermentative lactobacillus (42%) following running purification and biochemial tests of arginine hydrolysis and fermentation of carbohydrates and co2 production from glucose. Based on simillarity biochemical and growth characteristics the 36 isolated lactobacillus were classified into 15 groups and 3 stories. All the 15 isolated groups of lactobacillus and Lactobacillus plantarum (standard strain) were sensitive to Thymus vulgaris essential oil based on the characteristics of colonies. More in vivo experiments are required for reaching to the practical application of Thymus vulgaris essential oil utilization against the involved lactobacillus with rumen acidosis.