Goitrin and thiouracil belong to the thyreostats, a group of compounds that inhibit the thyroid function; thyreostats can be divided into two main groups: naturally occurring compounds, mainly originated from glucosinolates, and xenobiotic (thyreostatic drugs) compounds, used as growth-promoting agents and performance enhancers in food-producing animals. Recently, thiouracil was detected at low levels in urine and cow milk samples and a natural origin due to ingestion of Brassicaceae-rich feed was demonstrated. Considering that the natural occurrence of both goitrin and thiouracil can be due to the same type of feeding for cattle, a liquid chromatography–tandem mass spectrometry (LC-MS/ MS) method for their simultaneous determination at trace levels in milk and cheese was developed. Then, a limited survey and a study to evaluate their carry-over from milk to curd and ripened cheese were carried out. For both compounds, the limit of detection (LOD) and the limit of quantification (LOQ) were 0.07 and 0.20 μg L−1 in milk, and 0.05 and 0.16 μg kg−1 in cheese; the average recovery values were above 80 %. In milk, their incidence was very high (≥95 %); the maximum value was 3.6 and 8.7 μg L−1 for goitrin and thiouracil, respectively. In cheese, only thiouracil was detected at levels close to those in milk. During cheese-making, both compounds remained mainly in the whey; during ageing, goitrin content decreased quickly and it was never detected after 2 months of ripening.