The results showed that the key action targets of oregano essential oil in the treatment of dairy bovine mastitis were TNF, TLR4, ALB, IL-1β, TLR2, IL-6, IFNG, and MyD88, and the main signaling pathways were PI3K-Akt, MAPK, IL-17, NF-κ B. The molecular docking confirmed that thymol and carvacrol in oregano essential oil had a good binding ability to the core targets of TNF, IL-6, MyD88, and ALB. This evidence concerns the gene TLR4 and mastitis.