The aim of the study was to investigate serum and milk concentrations of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), and indoleamine 2,3-dioxygenase (IDO) activity in cows suffering from subclinical mastitis caused by coagulase-negative staphylococci (MSCNS). TRP and kynurenines were determined by high-performance liquid chromatography (HPLC), and IDO activity was calculated as the KYN/TRP ratio. The blood and milk samples were collected from 40 midlactation Holstein-Fresian cows from two herds in the Lublin region in Poland. In the milk samples from 20 cows with subclinical mastitis, coagulase-negative staphylococci were isolated and in the milk obtained from healthy cows growth of microorganisms was not detected. TRP, KYN and KYNA concentrations were significantly lower in milk of cows with MSCNS compared to control animals (4.47 vs. 7.24 µM, 0.14 vs. 0.21 µM, 1.58 vs. 2.18 nM, respectively). There was no statistically significant difference in TRP and KYNA concentrations in serum between the studied animal groups (32.97 vs. 39.29 µM, 31.3 vs. 26.5 nM, respectively). In turn, the level of KYN was lower in the serum (0.81 vs. 1.13 µM) of cows with mastitis compared to healthy ones. No statistically significant differences in IDO activity, both in serum and in milk (25.24 and 27.55, 28.56 and 27.17, respectively) was revealed between the studied groups. These findings may have potential implications for diagnosis of mastitis in cows because reduction of these parameters in milk might be a marker predicting the occurrence of the disease.