Biomonitoring of mycotoxins and their metabolites in biological fluids is increasingly used to assess human exposure. In this study, biomarkers of ochratoxin A (OTA) and citrinin (CIT) exposure were determined in a large number of serum samples from healthy blood donors in Switzerland. In 2019, 700 samples from different regions were obtained. From 240 donors, a second sample (taken 2-9 months later) was available for analysis. Moreover, 355 blood donor samples from 2005 from all regions in Switzerland and 151 additional samples from the southern Swiss region of Ticino from 2005 could be analysed.OTA, 2'R-ochratoxin A (2'R-OTA), ochratoxin alpha (OTα), CIT and dihydrocitrinone (DH-CIT) were analysed using validated targeted methods including precipitation and online SPE clean-up.OTA and 2'R-OTA were frequently detected (OTA in 99%; 2'R-OTA in 51% of the tested samples). The mean concentration in all positive samples was 0.4 ng/mL for OTA and 0.2 ng/mL for 2'R-OTA. OTα was not detected in any sample above the limit of quantification (LOQ). In contrast to OTA, CIT and DH-CIT were only quantifiable in 2% and 0.1% of the samples, respectively. No significant trend was observed between the samples from 2005 and the more recent samples, but OTA concentrations were usually higher in serum samples from the southern Swiss region of Ticino and in males compared to females.Our extensive data fit well within the framework of previously published values for the healthy adult European population.