Ochratoxin (OT) A is a toxin produced by several species of fungi, including Aspergillus and Penicillium, and can contaminate various food and feedstuffs. Its hepatotoxic, nephrotoxic, and teratogenic effects in animals are well documented. Ruminants possess physiological (i.e., microbial) systems for detoxifying mycotoxins. This experiment investigated the transmission of OTA and its main metabolite OTα into plasma, serum, milk, urine and faeces in lactating Simmental cows. The animals were fed with OTA-containing concentrate-rich diets composed of grass silage, corn silage and concentrate at 37.1%, 12.5% and 50.4% on dry matter basis, respectively. Twelve cows were randomly assigned to two equal groups (OTA-Low/OTA-High), which received 250 and 2500 µg/kg DM diet for seven days, respectively. The concentrations of OTA and OTα in the mentioned biological matrices were measured via high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). In addition, daily feed intake, chewing behaviour, milk production and clinical parameters were evaluated daily. Milk composition, haematological parameters and hepatic enzymes were evaluated on days 0 and 7 of the experiment. Using three cows per group, the analytical method was validated by determining the following limit of quantification (LOQs) for OTA and OTα: In plasma, serum, and milk 0.5 ng/mL, urine diluted to 10 mM creatinine 5 ppb and in dried faeces 50 ng/g. All stated LOQs are applicable for both analytes. On day 7, the average (± SD) concentrations of OTA in plasma, serum, milk (ng/mL), urine (ppb diluted to 10 mM creatinine) and faeces (ng/g DM) in the group OTA-High were 8.1(±0.9), 7.0(±1.1), 0.0(±0.0), 9.9(±3.2), 222(±94.3), respectively, and for OTα, 18.3(±1.5), 24.8(± 5.0), 4.8(±1.4), 1271(± 609), 1523(±314), respectively. Taken together, ingesting concentrate-rich diets containing up to 2500 μg OTA/kg DM diet for seven days did not affect feed intake, milk production and composition, chewing behaviour, clinical and haematological parameters, or hepatic function of cows. Because most OTA was microbially metabolized, low concentrations were detected in plasma/serum, and carry-over into milk was not detected. The excretion was mainly as OTα in urine and faeces.