Tea tree oil (TTO) plays an important role in antibacterial activity and anti-inflammatory properties. However, effect of TTO on the growth of Staphylococcus aureus, bovine mammary epithelial cells (BMECs) and polymorphonuclear leukocytes (PMNL) function remains not reported. Therefore, the aim of study was to investigate the effects of TTO on the growth of S. aureus and expression of genes involved in inflammatory response in BMECs and PMNL. Statistical analysis was performed using the SPSS 16.0 software. P < 0.05 was considered significant. Our data demonstrated that addition of 0.025 and 0.05% TTO enhanced (P < 0.05) the viability of BMECs in culture, whereas 0.1% TTO inhibited (P < 0.05) the viability of BMECs. The BMECs treated by 0.0125, 0.025, and 0.05% TTO exposed to S. aureus promoted (P < 0.05) the viability of BMECs exposed to S. aureus. Meanwhile, the growth of S. aureus was significantly inhibited (P < 0.001) after treatment with TTO after 4 h. In addition, the rate of S. aureus invasion into BMECs was also attenuated (P < 0.05) by 0.05% of TTO compared with controls. The morphology of S. aureus has a profound change by the treatment of 0.05% TTO using scanning electron microscope compared with controls. The adding of 0.05% TTO decreased (P < 0.05) the levels of mRNA encoding TNF-α and IL-1β in S. aureus-exposed BMECs compared with S. aureus-exposed BMEC, but chemokine IL-8 was upregulated (P < 0.05). Moreover, the addition of 0.05% TTO enhanced (P < 0.05) the expression level of β-casein in S. aureus-exposed mammary gland explants compared with S. aureus-exposed mammary gland explants. The PMNL incubated with 0.00625, 0.0125, 0.05% TTO had no inhibitory effect on PMNL viability. The expression of IL-1β, IL-6, IL-8, TLR-2, TLR-4, L-SELETIN, IRAKII, TRAF-6, Lysozyme, SOD2 were not altered by incubation of PMNL with 0.05% TTO for 6 h. However, the PMNL incubated with 0.05% TTO for 6 h reduced (P < 0.01) the expression level of IL-10, TNF-α, and NFKBIA relative to the control groups. These findings revealed that the use of TTO may be a potent therapeutic tool against bovine mastitis caused by S. aureus and thereby mediate protective immunity.