Treatment of mastitis induced by Staphylococcus aureus is notoriously difficult since S. aureus may rapidly develop biofilms and resistance to most antibiotics, including cefquinome (Cef). This study aimed to investigate whether zinc oxide nanoparticles (ZnONPs) would ameliorate cefquinome (Cef) side effects on buffalo mammary gland epithelial (MGE) cells and increase its antibacterial potential. We first evaluated the antibacterial impact of ZnONPs and/or Cef and found significantly higher antibacterial potential for the combined therapy against Staphylococcus aureus isolated from buffalo mastitic milk than individual therapy. Next, we used the MTT assay to evaluate the cytotoxic effects of ZnONPs and/or Cef on MGE cells and found significantly lowered cytotoxic effects for the combined therapy compared to individual therapy with Cef alone. To detect the associated molecular pathway, we applied qPCR and found that the combined therapy with ZnONPs and Cef significantly downregulated apoptosis-related genes (Bax and caspase 3), inflammation-associated genes (IL1β and TNFα), and significantly upregulated the anti-apoptotic Bcl2 gene in MGE cells compared with individual therapy. Finally, oxidative stress was decreased in MGE cells after co-treatment with ZnONPs and Cef, as shown by increased expression of the NrF2 and HO-1 genes, decreased levels of MDA, and increased levels of CAT, SOD, and GPx. These findings led us to the conclusion that ZnONP treatment not only reduced the negative effects of Cef on MGE cells, but also boosted the antibiotic's efficacy. Therefore, ZnONPs should be used in conjunction with Cef to treat mastitis especially that caused by Staphylococcus aureus.