Introduction The aim of the study was evaluation of the concentrations of interleukin (IL)-1β, IL-8, IL-12β and tumour necrosis factor alpha (TNF-α) in the serum and milk of cows with mastitis caused by Streptococcus agalactiae. Material and Methods A total of 60 milk samples from diseased cows and 30 milk samples from healthy cows were included in the study. Blood and milk samples were taken from Holstein-Friesian cows from three herds (two in tie-stall and one in a free-stall housing system) in Lublin Province in Poland. The concentrations of cytokines in blood serum and quarter milk samples were determined by ELISA. Results The levels of IL-1β, IL-8, IL-12β and TNF-α were significantly higher in the milk of cows suffering from mastitis caused by S. agalactiae compared to the milk of healthy cows (263.03 vs 55.36 pg/mL, 298.34 vs 131.82 pg/mL, 604.10 vs 139.17 pg/mL and 460.86 vs 78.82 pg/mL, respectively). In the group of sick cows, cytokine levels were significantly higher in milk than in serum (263.03 vs 55.25 pg/mL for IL-1β, 298.34 vs 164.22 pg/mL for IL-8, 604.10 vs 70.34 pg/mL for IL-12β and 460.86 vs 104.78 pg/mL for TNF-α). Conclusion The results confirm the involvement of the entire bovine immune system to protect against the bacteria first locally in the udder. The response of the mammary gland to infection caused by S. agalactiae is rapid and already very strong at the beginning of the infection.