Mastitis is a frequent disease in modern dairy cows, but ancient cattle breeds seem to be naturally more resistant to it. Primary bovine mammary epithelial cells from the ancient Highland and White Park (n = 5) cattle and the modern dairy breeds Brown Swiss and Red Holstein (n = 6) were non-invasively isolated from milk, cultured, and stimulated with the heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus to compare the innate immune response in vitro. With reverse transcription quantitative polymerase chain reaction (RT-qPCR), the breeds differed in the basal expression of 16 genes. Notably CASP8, CXCL8, Toll-like receptors 2 and 4 (TLR2 and TLR4) expression were higher in the ancient breeds (P < 0.05). In the modern breeds, more genes were regulated after stimulation. Breed differences (P < 0.05) were detected in C3, CASP8, CCL2, CD14, LY96 and transforming growth factor β1 (TGFβ1) regulation. Principal component analysis separated the ancient from the modern breeds in their basal expression, but not after stimulation. ELISA of lactoferrin and serum amyloid A protein revealed breed differences in control and S. aureus treated levels. The immune reaction of ancient breeds seemed less intensive because of a higher basal expression, which has been shown before to be beneficial for the animal. For the first time, the innate immune response of these ancient breeds was studied. Previous evidence of breed and animal variation in innate immunity was confirmed.