On account of the pivotal role of herbal compounds in all scientific research, this study aimed to detect the effect of garlic essential oil nanoemulsion (GON) on Trueperella pyogenes (T. pyogenes). Four isolates of T. pyogenes were grown on brain heart infusion agar were used for extraction of the bacterial DNA for detection of 16 SrRNA and rpoB genes. The physico-chemical characteristics of GON were analyzed through GC-mass analysis, and Nanotrac- Zeta sizer. The antibacterial activity was studied by minimum inhibitory concentration using the transmission electron microscope. All isolates used were positive for16 SrRNA and rpoB genes. The average Garlic oil nanoemulsion size was 118 nm with major homogeneity. Garlic essential oil nanoemulsion has a negative charge with automatic polarity with 89 us/cm conductivity. Garlic essential oil nanoemulsion was safe to be used and the fatty acids, especially oleic and stearic acids, were its major particulars. Garlic oil nanoemulsion has deleterious effects on T. pyogenes as causes cell lysis, cytoplasmic leakage, and nanoparticle clusters on the bacterial cell. Furthermore, it resulted in alterations to the exterior surfaces, interior characteristics and biological activity of the cells, so it should be studied on a large scale and trial to explore in vivo efficacy of this product should be done.