AbstractSubclinical mastitis by Staphylococcus aureus (SAU) and by non-aureus staphylococci (NAS) is a major issue in the water buffalo. To understand its impact on milk, 6 quarter samples with >3,000,000 cells/mL (3 SAU-positive and 3 NAS-positive) and 6 culture-negative quarter samples with <50,000 cells/mL were investigated by shotgun proteomics and label-free quantitation. A total of 1530 proteins were identified, of which 152 were significantly changed. SAU was more impacting, with 162 vs 127 differential proteins and higher abundance changes (P < 0.0005). The 119 increased proteins had mostly structural (n = 43, 28.29%) or innate immune defence functions (n = 39, 25.66%) and included vimentin, cathelicidins, histones, S100 and neutrophil granule proteins, haptoglobin, and lysozyme. The 33 decreased proteins were mainly involved in lipid metabolism (n = 13, 59.10%) and included butyrophilin, xanthine dehydrogenase/oxidase, and lipid biosynthetic enzymes. The same biological processes were significantly affected also upon STRING analysis. Cathelicidins were the most increased family, as confirmed by western immunoblotting, with a stronger reactivity in SAU mastitis. S100A8 and haptoglobin were also validated by western immunoblotting. In conclusion, we generated a detailed buffalo milk protein dataset and defined the changes occurring in SAU and NAS mastitis, with potential for improving detection (ProteomeXchange identifier PXD012355).